Inhibition of HIV-1 replication by disruption of the processing of the viral capsid-spacer peptide 1 protein

ABSTRACT

Inhibition of HIV-1 replication by disrupting the processing of the viral Gag capsid (CA) protein (p24) from the CA-spacer peptide 1 (SP1) protein precursor (p25) is disclosed. Amino acid sequences containing a mutation in the Gag p25 protein, with the mutation resulting in a decrease in the inhibition of processing of p25 to p24 by dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, polynucleotides encoding such mutated sequences and antibodies that selectively bind such mutated sequences are also included. Methods of inhibiting, inhibitory compounds and methods of discovering inhibitory compounds that target proteolytic processing of the HIV Gag protein are included. In one embodiment, such compounds inhibit the interaction of the HIV protease enzyme with Gag by binding to the Gag proteolytic cleavage site rather than to the protease enzyme. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the Gag proteolytic cleavage site can be used in screening assays to identify compounds that target proteolytic processing.

RELATED U.S. APPLICATION DATA

[0001] This application claims priority to U.S. Provisional ApplicationNos. 60/496,660, filed Aug. 21, 2003, and 60/443,180, filed Jan. 29,2003, both applications herein incorporated by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0002] The U.S. Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms of GrantNo. 2R44AI051047-02 awarded by NIH/NIAID.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention includes methods of inhibiting, inhibitors andmethods of discovery of inhibitors of HIV infection.

[0005] 2. Background

[0006] Human Immunodeficiency Virus (HIV) is a member of thelentiviruses, a subfamily of retroviruses. Many retroviruses arewell-known carcinogens. HIV per se is not known to cause cancer inhumans or other animals, but it does present a formidable challenge tothe host. The viral genome contains many regulatory elements which allowthe virus to control its rate of replication in both resting anddividing cells. Most importantly, HIV infects and invades cells of theimmune system; it breaks down the body's immune system and renders thepatient susceptible to opportunistic infections and neoplasms. Theimmune defect appears to be progressive and irreversible, with a highmortality rate that approaches 100% over several years.

[0007] HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of theimmune system which express the cell surface differentiation antigenCD4, also known as OKT4, T4 and leu3. The viral tropism is due to theinteractions between the viral envelope glycoprotein, gp120, and thecell-surface CD4 molecules (Dalgleish et al., Nature 312:763-767(1984)). These interactions not only mediate the infection ofsusceptible cells by HIV, but are also responsible for the virus-inducedfusion of infected and uninfected T cells. This cell fusion results inthe formation of giant multinucleated syncytia, cell death, andprogressive depletion of CD4 cells in HIV-infected patients. Theseevents result in HIV-induced immunosuppression and its subsequentsequelae, opportunistic infections and neoplasms.

[0008] In addition to CD4⁺ T cells, the host range of HIV includes cellsof the mononuclear phagocytic lineage (Dalgleish et al., supra),including blood monocytes, tissue macrophages, Langerhans cells of theskin and dendritic reticulum cells within lymph nodes. HIV is alsoneurotropic, capable of infecting monocytes and macrophages in thecentral nervous system causing severe neurologic damage. Macrophage andmonocytes are major reservoirs of HIV. They can interact and fuse withCD4-bearing T cells, causing T cell depletion and thus contributing tothe pathogenesis of AIDS.

[0009] Considerable progress has been made in the development of drugsfor HIV-1 therapy. Therapeutic agents for HIV can include, but not arenot limited to, at least one of AZT, 3TC, ddC, d4T, ddI, tenofovir,abacavir, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir,indinavir, nelfinavir, lopinavir and amprenavir, or any otherantiretroviral drugs or antibodies in combination with each other, orassociated with a biologically based therapeutic, such as, for example,gp41-derived peptides enfuvirtide (Fuzeon; Timeris-Roche) and T-1249(Trimeris), or soluble CD4, antibodies to CD4, and conjugates of CD4 oranti-CD4, or as additionally presented herein. Combinations of thesedrugs are particularly effective and can reduce levels of viral RNA toundetectable levels in the plasma and slow the development of viralresistance, with resulting improvements in patient health and life span.

[0010] Despite these advances, there are still problems with thecurrently available drug regimens. Many of the drugs exhibit severetoxicities, have other side-effects (e.g., fat redistribution) orrequire complicated dosing schedules that reduce compliance and therebylimit efficacy. Resistant strains of HIV often appear over extendedperiods of time even on combination therapy. The high cost of thesedrugs is also a limitation to their widespread use, especially outsideof developed countries.

[0011] There is still a major need for the development of additionaldrugs to circumvent these issues. Ideally these would target differentstages in the viral life cycle, adding to the armamentarium forcombination therapy, and exhibit minimal toxicity, yet have lowermanufacturing costs.

[0012] HIV virion assembly takes place at the surface membrane of theinfected cell where the viral Gag polyprotein accumulates, leading tothe assembly of immature virions that bud from the cell surface. Withinthe virion, Gag is cleaved by the viral proteinase (PR) into the matrix(MA), capsid (CA), nucleocapsid (NC), and C-terminal p6 structuralproteins (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)). Gagprocessing induces a reorganization of the internal virion structure, aprocess termed “maturation.” In mature HIV particles, MA lines the innersurface of the membrane, while CA forms the conical core which encasesthe genomic RNA that is complexed with NC. Cleavage and maturation arenot required for particle formation but are essential for infectivity(Kohl, N. et al., Proc. Natl. Acad. Sci. USA 85:4686-4690, (1998)).

[0013] CA and NC as well as NC and p6 are separated on the Gagpolyprotein by short spacer peptides of 14 and 10 amino acids (p2),respectively (spacer peptide 1 (SP1) and SP2, respectively) (Wiegers K.et al., J. Virol. 72:2846-2854 (1998), Pettit, S. C. et al., J. Virol.68:8017-8027 (1994), Liang et al. J. Virol. 76:11729-11737 (2002)).These spacer peptides are released by PR-mediated cleavages at their Nand C termini during particle maturation. The individual cleavage siteson the HIV Gag and Gag-Pol polyproteins are processed at different ratesand this sequential processing results in Gag intermediates appearingtransiently before the final products. Such intermediates may beimportant for virion morphogenesis or maturation but do not contributeto the structure of the mature viral particle (Weigers et al. andPettit, et al., supra). The initial Gag cleavage event occurs at the Cterminus of SP1 and separates an N-terminal MA-CA-SP1 intermediate froma C-terminal NC-SP2-p6 intermediate. Subsequent cleavages separating MAfrom CA-SP1 and NC-SP2 from p6 occur at an approximately 10-fold-lowerrate. Cleavage of SP1 from the C terminus of CA is a late event andoccurs at a 400-fold-lower rate than cleavage at the SP1-NC site(Weigers et al. and Pettit, et al., supra). The uncleaved CA-SP1intermediate protein is alternatively termed “p25,” whereas the cleavedCA protein is termed “p24.”

[0014] Cleavage of SP1 from the C terminus of CA appears to be one ofthe last events in the Gag processing cascade and is required for finalcapsid condensation and formation of mature, infectious viral particles.Electron micrographs of mature virions reveal particles having electrondense conical cores. On the other hand, electron microscopy studies ofviral particles defective for CA-SP1 cleavage show particles having aspherical electron-dense ribonucleoprotein core and a crescent-shaped,electron-dense layer located just inside the viral membrane (Weigers etal., supra). Mutations at or near the CA-SP1 cleavage site have beenshown inhibit Gag processing and disrupt the normal maturation process,thereby resulting in the production of non-infectious viral particles(Weigers et al., supra). Phenotypically, these particles exhibit adefect in Gag processing (which manifests itself in the presence of ap25 (CA-SP1) band in Western blot analysis) and the aberrant particlemorphology described above which results from defective capsidcondensation.

[0015] Previously, betulinic acid and platanic acid were isolated fromSyzigium claviflorum and were determined to have anti-HIV activity.Betulinic acid and platanic acid exhibited inhibitory activity againstHIV-1 replication in H9 lymphocyte cells with EC₅₀ values of 1.4 μM and6.5 μM, respectively, and therapeutic index (T.I.) values of 9.3 and 14,respectively. Hydrogenation of betulinic acid yielded dihydrobetulinicacid, which showed slightly more potent anti-HIV activity with an EC₅₀value of 0.9 and a T.I. value of 14 (Fujioka, T., et al., J. Nat. Prod.57:243-247 (1994)). Esterification of betulinic acid with certainsubstituted acyl groups, such as 3′,3′-dimethylglutaryl and3′,3′-dimethylsuccinyl groups produced derivatives having enhancedactivity (Kashiwada, Y., et al., J. Med. Chem. 39:1016-1017 (1996)).Acylated betulinic acid and dihydrobetulinic acid derivatives that arepotent anti-HIV agents are also described in U.S. Pat. No. 5,679,828.Anti-HIV assays indicated that 3-O-(3′,3′-dimethylsuccinyl)-betulinicacid and the dihydrobetulinic acid analog both demonstrated extremelypotent anti-HIV activity in acutely infected H9 lymphocytes with EC₅₀values of less than 1.7×10⁻⁵ μM, respectively. These compounds exhibitedremarkable T.I. values of more than 970,000 and more than 400,000,respectively.

[0016] U.S. Pat. No. 5,468,888 discloses 28-amido derivatives of lupanes

[0017] R=H (Betulinic acid) that are described as having acytoprotecting effect for HIV-infected cells.

[0018] Japanese Patent Application No. JP 01 143,832 discloses thatbetulin and 3,28-diesters thereof are useful in the anti-cancer field.

[0019] U.S. Pat. No. 6,172,110 discloses betulinic acid anddihydrobetulin derivatives which have the following formulae orpharmaceutically acceptable salts thereof,

[0020] Betulin and Dihydrobetulin Derivatives

[0021] wherein R₁ is a C₂-C₂₀ substituted or unsubstituted carboxyacyl,R₂ is a C₂-C₂₀ substituted or unsubstituted carboxyacyl; and R₃ ishydrogen, halogen, amino, optionally substituted mono- or di-alkylamino,or —OR₄, where R₄ is hydrogen, C₁₋₄ alkanoyl, benzoyl, or C₂-C₂₀substituted or unsubstituted carboxyacyl; wherein the dashed linerepresents an optional double bond between C20 and C29.

[0022] U.S. patent application Ser. No. 60/413,451 discloses3,3-dimethylsuccinyl betulin and is herein incorporated by reference.Zhu, Y-M. et al., Bioorg. Chem Lett. 11:3115-3118 (2001); Kashiwada Y.et al., J. Nat. Prod. 61:1090-1095 (1998); Kashiwada Y. et al., J. Nat.Prod. 63:1619-1622 (2000); and Kashiwada Y. et al., Chem. Pharm. Bull.48:1387-1390 (2000) disclose dimethylsuccinyl betulinic acid anddimethylsuccinyl oleanolic acid. Esterification of the 3′ carbon ofbetulin with succinic acid produced a compound capable of inhibitingHIV-1 activity (Pokrovskii, A. G. et al., Gos. Nauchnyi Tsentr Virusol.Biotekhnol. “Vector, ” 9:485-491 (2001)).

[0023] Published International Application No. WO 02/26761 discloses theuse of betulin and analogs thereof for treating fungal infections.

[0024] There exists a need for new HIV inhibition methods that areeffective against drug resistant strains of the virus. The strategy ofthis invention is to provide therapeutic methods and compounds thatinhibit the virus in different ways from approved therapies.

[0025] The compound and methods of the present invention have a novelmechanism of action and therefore are active against HIV strains thatare resistant to current reverse transcriptase and protease inhibitors.As such, this invention offers a completely new approach for treatingHIV/AIDS.

BRIEF SUMMARY OF THE INVENTION

[0026] Generally, the invention provides methods of inhibiting,inhibitory compounds and methods of identifying inhibitory compoundsthat target proteolytic processing of the HIV-1 Gag protein. In oneembodiment, such compounds inhibit the interaction of a protease enzymewith HIV-1 Gag protein. In another embodiment, such inhibition ofinteraction occurs via the binding of a compound to Gag. The inhibitionof protease cleavage of the CA-SP1 protein of HIV-1 Gag by3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB) is one example, butother proteolytic cleavage sites can be targeted by a similar approachusing inhibitory compounds that interact with the substrate in a mannersimilar to that in which DSB interacts with Gag.

[0027] A first aspect of the invention is directed to a method ofinhibiting the processing of the viral Gag p25 protein (CA-SP1) to p24(CA), but having no effect on other Gag processing steps.

[0028] A second aspect of the invention is directed to a method foridentifying compounds that inhibit processing of the viral Gag p25protein (CA-SP1) to p24 (CA), but have no effect on other Gag processingsteps.

[0029] A third aspect of the invention is drawn to a compound orpharmaceutical composition identified by the method for identifyingcompounds that inhibit HIV-1 replication disclosed herein.

[0030] A fourth aspect of the present invention is directed to apolynucleotide comprising a sequence which encodes an amino acidsequence containing a mutation in the Gag p25 protein, said mutationresulting in a decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl) betulinic acid. This aspect of theinvention is also directed to a vector, virus and host cell comprisingsaid polynucleotide, and a method of making said protein.

[0031] A fifth aspect of the present invention is directed to an aminoacid sequence containing a mutation in the Gag p25 protein, saidmutation resulting in a decrease in the inhibition of processing of p25to p24 by 3-O-(3′,3′-dimethylsuccinyl) betulinic acid.

[0032] A sixth aspect of the invention is directed to an antibody whichselectively binds an amino acid sequence containing a mutation in theGag p25 protein, said mutation resulting in a decrease in the inhibitionof processing of p25 to p24 by 3-O-(3′,3′-dimethylsuccinyl) betulinicacid. Also included in this aspect of the invention are a method ofmaking said antibody, a hybridoma producing said antibody and a methodof making said hybridoma.

[0033] A seventh aspect of the invention is directed to a kit comprisinga polynucleotide, polypeptide or antibody disclosed herein.

[0034] The invention further relates to a method of inhibiting HIV-1infection in cells of an animal by contacting said cells with a compoundthat blocks the maturation of virus particles released from treatedinfected cells. In one embodiment, the released virus particles exhibitnon-condensed cores and a distinctive thin electron-dense layer near theviral membrane and have reduced infectivity. A method is included ofcontacting animal cells with a compound that both inhibits processing ofthe viral Gag p25 protein and that disrupts the maturation of virusparticles. Also, included is a method of treating HIV-infected cells,wherein the HIV infecting said cells does not respond to other HIVtherapies.

[0035] This invention further includes a method for identifyingcompounds that inhibit processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), but have no significant effect on other Gag processingsteps. The method involves contacting HIV-1 infected cells with a testcompound, and thereafter analyzing virus particles that are released todetect the presence of p25. Methods to detect p25 include westernblotting of viral proteins and detecting using an antibody to p25, gelelectrophoresis, and imaging of metabolically labeled proteins. Methodsto detect p25 also include immunoassays using an antibody to p25 or SP1to distinguish p25 from p24. For example, a microwell assay can beperformed where p25 in detergent-solubilized virus is captured using anantibody specific for SP1 that is bound to the plastic microwell plate.Following a washing step, bound p25 is detected using an antibody to p24that is conjugated to an appropriate detection reagent (e.g. alkalinephosphatase for an enzyme-linked immunosorbent assay). Virus released bycells treated with compounds that act via this mechanism will haveincreased levels of p25 compared with untreated virions.

[0036] The invention is further directed to a method for identifyingcompounds involving contacting HIV-1 infected cells with a compound, andthereafter analyzing virus particles released by the contacted cells, bythin-sectioning and transmission electron microscopy, and identifying ifvirion particles are detected with non-condensed cores and a distinctivethin electron-dense layer near the viral membrane.

[0037] The invention is also directed to compounds identified by theaforementioned screening methods.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0038]FIG. 1. DSB does not disrupt the activity of HIV-1 protease at aconcentration of 50 μg/mL. In DSB-containing samples recombinant Gag isprocessed correctly. In contrast, indinavir blocks protease activity at5 μg/mL as evidenced by the absence of bands corresponding to p24 andthe MA-CA precursor.

[0039]FIG. 2. Western blots of virion-associated Gag derived fromchronically infected H9/HIV-1_(IIIB), H9/HIV-2_(ROD), andH9/SIV_(mac251) in the presence of DSB (1 μg/mL), indinavir (1 μg/mL) orcontrol (DMSO). Gag proteins were visualized using HIV-Ig (HIV-1) ormonkey anti-SIV_(mac251) serum (HIV-2 and SIV; NIH AIDS Research andReference Reagent Program).

[0040]FIG. 3. EM analysis of DSB-treated HIV-1 infected cells. The EMdata show two primary differences between DSB-treated and untreatedsamples. Virions generated in the presence of DSB are characterized byan absence of conical, mature cores. In these samples the cores areuniformly spherical and often acentric. Secondly, many virions displayan electron dense layer inside the lipid bilayer but outside the core(indicated with arrows in the DSB-treated sample panels). In theDSB-treated samples no mature viral particles were observed.

[0041]FIG. 4 depicts a amino acid sequences in the region of the CA-SP1cleavage site from DSB-sensitive HIV-1 isolates NL4-3 and RF (#1; SEQ IDNO: 1) and DSB-resistant HIV-1 isolates (#2; SEQ ID NO: 2 (NL4-3), and#3; SEQ ID NO: 3 (RF)). The differences between the native andDSB-resistant sequences involve an alanine to valine change at the firstdownstream residue (#2) and an alanine to valine change in the thirddownstream residue (#3) from the CA-SP1 cleavage site (−|−). Theseresidues are underlined and bolded for ease of identification.

[0042]FIG. 5 depicts the + sense consensus sequence for the A364VDSB-resistant NL4-3 mutant (SEQ ID NO: 4) beginning with the start ofthe gag coding sequence and continuing into pol, including the entireprotease coding region. Missense mutations not found in the wild-typeNL4-3 GENBANK M19921 sequence are in bold and gray shadowing. The codingsequence for the consensus CA-SP1 cleavage site is underlined. Theshaded area including the cleavage site denotes the SP1 sequence. Thefirst mutation is the A364V mutation; the second amino acid difference(in protease) was also found in the parental clone and has beenconfirmed to correspond to a sequencing error in the original GENBANKentry. Therefore, no mutations actually occurred in protease.

[0043]FIG. 6 depicts the + sense consensus sequence for theDSB-sensitive NL4-3 parental isolate (SEQ ID NO: 5) that was passaged inthe absence of drug in parallel with the A364V mutant isolate.

[0044]FIG. 7 depicts the + sense consensus sequence for the A366VDSB-resistant HIV-1_(RF) mutant (SEQ ID NO: 6) beginning with the startof the Gag coding sequence and continuing through all of the codingsequence for Pro and part of RT. Missense mutations not found in thewild-type HIV-1_(RF) GENBANK M17451 sequence are shadowed in gray. TheCA-SP1 cleavage site is underlined. The only missense mutation not alsofound in the identically passaged DSB-sensitive isolate is the A366Vmutation in the CA-SP1 cleavage site.

[0045]FIG. 8 depicts the + sense consensus sequence for theDSB-sensitive HIV-1_(RF) parental isolate (SEQ ID NO: 7), that waspassaged in the absence of drug in parallel with the A366V mutantisolate.

[0046]FIG. 9 depicts the polynucleotide sequences, SEQ ID NO: 8 and SEQID NO: 9, which encode the polypeptides designated herein as SEQ ID NO:2 and SEQ ID NO: 3, respectively. SEQ ID NO: 10 depicts the nucleotidesequence that encodes the parental polypeptide sequence designatedherein as SEQ ID NO: 1.

[0047]FIG. 10 depicts the amino acid sequence from SIV_(mac239) in theregion of the CA-SP1 cleavage site (−|−) (SEQ ID NO: 11).

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention is directed to methods of inhibiting HIV-1replication in the cells of an animal that involve using compounds thatdisrupt the processing of the viral Gag p25 protein (CA-SP1) to the p24protein (CA), thereby resulting in the formation of non-infectious viralparticles.

[0049] Mutant viruses defective in CA-SP1 cleavage have been shown to benon-infectious (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)).3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB) is an example of acompound that disrupts p25 to p24 processing and potently inhibits HIV-1replication. This compound's activity is specific for the p25 to p24processing step, not other steps in Gag processing. Furthermore, DSBtreatment results in the aberrant HIV particle morphology as describedin FIG. 3.

[0050] Mutant forms of HIV-1 have been generated in which the SP1sequence is modified making these strains resistant to compounds thatdisrupt CA-SP1 processing. Data on these mutant viruses have been usedto identify the amino acid residues in native Gag that are implicated inthe antiviral activity of these compounds. In one embodiment, compoundsthat disrupt CA-SP1 processing inhibit the interaction of HIV-1 proteasewith the region of the Gag protein containing these amino acid residues.In another embodiment, compounds that disrupt CA-SP1 processing bind tothe region containing these amino acid residues. In another embodiment,compounds that disrupt CA-SP1 processing bind to another region of Gagand thereby inhibit the interaction of HIV-1 protease with the region ofthe CA-SP1 cleavage site. In another embodiment, viruses or recombinantproteins that contain mutations in the region of the CA-SP1 cleavagesite can be used in screening assays to identify compounds that disruptCA-S P1 processing.

[0051] Amino acid residues in HIV-1 Gag that are involved in thedisruption of CA-SP1 processing by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB) were identified by sequencing the Gag-Pol gene ofvirus isolates that had been selected for resistance to DSB. The aminoacid sequences from these resistant viruses were compared with theGag-Pol gene sequences from DSB-sensitive HIV-1 isolates. Two singleamino acid changes were identified in the DSB-resistant viruses, analanine (Ala) to valine (Val) substitution at residue 364 (SEQ ID NO: 4)and in a second isolate, at residue 366 (SEQ ID NO: 6), in the Gagpolyprotein (see FIG. 4). These residues are located immediatelydownstream of the CA-SP1 cleavage site (at the N-terminus of SP1).Alanine is highly conserved at these positions throughout all HIV-1clades in the Los Alamos National Laboratory database. The five aminoacid residues upstream and downstream of the CA-SP1 cleavage site arealso highly conserved among the various clades. However, isoleucinereplaces leucine at the position one residue upstream of the cleavagesite in a number of clades (c.f., FIG. 4, SEQ ID NO. 1). (“HIV SequenceCompendium 2002,” Kuiken et al. eds. Los Alamos National Laboratory, LosAlamos, N.M.)

[0052] Structure of 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB)

[0053] The invention also includes a method of inhibiting HIV-1replication in cells of an animal comprising contacting infected cellswith a compound that inhibits the interaction of HIV protease withCA-SP1 which results in the inhibition of the processing of the viralGag p25 protein (CA-SP1) to p24 (CA), but has no significant effect onother Gag processing steps.

[0054] The invention is also drawn to a method of inhibiting HIV-1replication in cells of an animal comprising contacting infected cellswith a compound that inhibits processing of the viral Gag p25 protein(CA-SP1) to p24 (CA), thereby causing the viral particles that arereleased to be non-infectious, but has no significant effect on otherGag processing steps and/or wherein said inhibition does notsignificantly reduce the quantity of virus released from treated cellsand/or has no significant effect on the amount of RNA incorporation intothe released virions. The invention is also drawn to a method ofinhibiting HIV-1 replication in cells of an animal comprising contactinginfected cells with a compound that inhibits the maturation of virusparticles released from treated infected cells. In one embodiment, thesereleased viral particles exhibit spherical, electron-dense cores thatare acentric with respect to the viral particles, rather than theconical core structures associated with mature viral particles andpossess crescent-shaped, electron-dense layers lying just inside theviral membrane and have reduced or no infectivity. Some viral particlesmay also exhibit a conical core structure along with a preponderance ofthe viral particles that exhibit the altered core structure describedabove.

[0055] Abnormal p25 to p24 processing is also seen in other maturationbudding defects (Wild, C. T. et al., XIV Int. AIDS Conf., Barcelona,Spain, Abstract MoPeA3030 (July 2002)). These defects included mutationsin the Gag late domain (PTAP) or defects in TSG-101 mediated viralassembly that disrupt budding (Garrus, J. E et al., Cell, 107:55-65(2001) and Demirov, D. G. et al., J. Virology 76:105-117 (2002)).However, these mutations cause inhibition of virus release, while DSBtreatment does not have a significant effect on virus release. Themorphology of these maturation/budding mutants is also quite distinctfrom that observed following DSB-treatment. In addition, mutations thatinterfere with viral RNA dimerization and lead to the production ofimmature virus with defective core structures give a similar Gagprocessing phenotype (Liang, C. et al., J. Virology, 73:6147-6151,(1999)). However, in those cases RNA incorporation is inhibited and themorphology of particles released is distinct from those following DSBtreatment.

[0056] The method of inhibiting an HIV-1 replication in cells of ananimal disclosed herein includes a compound which binds near to or atthe site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA),thereby inhibiting the interaction of HIV protease with the CA-SP1cleavage site.

[0057] The invention includes any of the disclosed methods, wherein theHIV infecting said cells does not respond to other HIV therapies.

[0058] The present invention comprises a polynucleotide comprising asequence which encodes an amino acid sequence containing a mutation inthe HIV Gag p25 protein (CA-SP1), said mutation resulting in a decreasein the inhibition of processing of p25 (CA-SP1) to p24 (CA) by DSB. Thepolynucleotide of the invention includes a mutation which is optionallylocated near the CA-SP1 cleavage site or located in the SP1 region ofCA-SP1. Said mutation can be present in an amino acid sequence that isselected from the group consisting of KARVLVEAMS (SEQ ID NO: 2) orKARVIAEVMS (SEQ ID NO: 3). The polynucleotide of this invention is alsodrawn to sequences designated as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 or SEQ ID NO: 9. The invention also includes a vector comprising saidpolynucleotide, a host cell comprising said vector and a method ofproducing said polypeptides comprising incubating said host cell in amedium and recovering the polypeptide from the medium.

[0059] The invention further includes a polynucleotide that hybridizesunder stringent conditions to a polynucleotide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9.The invention also includes a polynucleotide which hybridizes to SEQ NO:5, SEQ ID NO: 7 or SEQ ID NO: 10, which contains a mutation whichresults in the decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl) betulinic acid, and also wherein saidmutation is optionally located in the SP1 region of CA-SP1. Theinvention is also directed to a vector comprising said polynucleotides,a host cell comprising said vector and a method of producing saidpolypeptides, comprising incubating said host cell in a medium andrecovering said polypeptide from the medium.

[0060] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both.Also, “isolated” nucleic acid molecule(s) of the invention is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0061] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritiated bases and unusual bases such as inosine.A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0062] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0063] “Mutant” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical mutant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the mutant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical mutant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A mutant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A mutant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a mutant that is notknown to occur naturally. Non-naturally occurring mutants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

[0064] Thus, the mutant, (or fragments, derivatives or analogs) of apolypeptide encoded by any one of the polynucleotides described hereinmay be (i) one in which at least one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue (aconserved amino acid residue(s), or at least one but less than tenconserved amino acid residues) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in which oneor more of the amino acid residues includes a substituent group, (iii)one in which the mature polypeptide is fused with another compound, suchas a compound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG:Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such mutants are deemed to be within the scope of those skilled in theart from the teachings herein. Polynucleotides encoding these mutantsare also encompassed by the invention. “Mutant” as used herein isequivalent to the term “variant.”

[0065] Substitutions of charged amino acids with another charged aminoacids and with neutral or negatively charged amino acids are included.Additionally, one or more of the amino acid residues of the polypeptidesof the invention (e.g., arginine and lysine residues) may be deleted orsubstituted with another residue to eliminate undesired processing byproteases such as, for example, furins or kexins. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)). Thus, the polypeptides of the presentinvention may include one or more amino acid substitutions, deletions oradditions, either from natural mutations or human manipulation.

[0066] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0067] The polynucleotides encompassed by this invention may have 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity with areference sequence, providing the reference polynucleotide encodes anamino acid sequence containing a mutation in the CA-SP1 protein, saidmutation which results in the decrease in the inhibition of processingof p25 to p24 by a 3-O-(3′,3′-dimethylsuccinyl) betulinic acid. Thepolynucleotides also encompassed by this invention include thosemutations which are “silent,” in which different codons encode the sameamino acid (wobble).

[0068] “Identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. The term “identity” is used interchangeablywith the word “homology” herein. In general, the sequences are alignedso that the highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans. Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to, those disclosed in Baxevanis and Oullette, Bioinformatics: APractical Guide to the Analysis of Genes and Proteins, Second Edition,Wiley-Interscience, New York, (2001). Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCS program package (Devereux, J. etal., Nucleic Acids Research 12(1):387, (1984)), BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J. Molec. Biol. 215:403, (1990)).

[0069] A polynucleotide having a nucleotide sequence having at least,for example, 95% “identity” to a reference nucleotide sequence isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

[0070] Similarly, by a polypeptide having an amino acid sequence havingat least, for example, 95% “identity” to a reference amino acidsequence, is intended that the amino acid sequence of the polypeptide isidentical to the reference sequence except that the polypeptide sequencemay include up to five amino acid alterations per each 100 amino acidsof the reference amino acid. To obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entirenucleotide sequence of any one of the nucleotide sequences of theinvention or any polynucleotide fragment (e.g., a polynucleotideencoding the amino acid sequence of the invention and/or C terminaldeletion).

[0071] Whether any particular nucleic acid molecule is at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences of the invention can be determined conventionallyusing known computer programs such as the BESTFIT program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,(Advances in Applied Mathematics 2:482-489 (1981)), to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, such thatthe percentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

[0072] In a specific embodiment, the identity between a sequence of thepresent invention and a subject sequence, also referred to as a globalsequence alignment, is determined using the FASTDB computer programbased on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). Preferred parameters used in a FASTDB alignment of DNAsequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0,Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 orthe length of the subject nucleotide sequence, whichever is shorter.According to this embodiment, if the subject sequence is shorter thanthe reference sequence because of 5′ or 3′ deletions, not because ofinternal deletions, a manual correction is made to the results to takeinto consideration the fact that the FASTDB program does not account for5′ and 3′ truncations of the subject sequence when calculating percentidentity. For subject sequences truncated at the 5′ or 3′ ends, relativeto the query sequence, the percent identity is corrected by calculatingthe number of bases of the query sequence that are 5′ and 3′ of thesubject sequence, which are not matched/aligned, as a percent of thetotal bases of the query sequence. A determination of whether anucleotide is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This correctedscore is what is used for the purposes of this embodiment. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score. For example, a 90 base subject sequence is aligned to a100 base query sequence to determine percent identity. The deletionsoccur at the 5′ end of the subject sequence and therefore, the FASTDBalignment does not show a matched/alignment of the first 10 bases at 5′end. The 10 unpaired bases represent 10% of the sequence (number ofbases at the 5′ and 3′ ends not matched/total number of bases in thequery sequence) so 10% is subtracted from the percent identity scorecalculated by the FASTDB program. If the remaining 90 bases wereperfectly matched the final percent identity would be 90%. In anotherexample, a 90 base subject sequence is compared with a 100 base querysequence. This time the deletions are internal deletions so that thereare no bases on the 5′ or 3′ of the subject sequence, which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Only bases 5′ and 3′ ofthe subject sequence which are not matched/aligned with the querysequence are manually corrected. No other manual corrections are madefor the purposes of this embodiment.

[0073] The present application is directed to nucleic acid molecules atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequence disclosed herein, or fragments thereof,irrespective of whether they encode a polypeptide having the disclosedfunctional activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having the disclosedfunctional activity, one of skill in the art would still know how to usethe nucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving the disclosed functional activity include, inter alia: (1)isolating the variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to determine cellular location or presenceof the disclosed sequences, and (3) Northern Blot analysis for detectingmRNA expression in specific tissues.

[0074] As used herein, the term “PCR” refers to the polymerase chainreaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202to Mullis et al., as well as improvements now known in the art. Inaccordance with the present invention there may be employed conventionalmolecular biology, microbiology, and recombinant DNA techniques withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Sambrook, J. and Russell, D. W. (2001)Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

[0075] The term “stringent conditions,” as used herein refers tohomology in hybridization, is based upon combined conditions of salt,temperature, organic solvents, and other parameters typically controlledin hybridization reactions, and well known in the art (Sambrook, et al.supra). The invention includes an isolated nucleic acid moleculecomprising, a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, thesequence complementary to the coding and/or noncoding (i.e.,transcribed, untranslated) sequence of any polynucleotide or apolynucleotide fragment as described herein. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising, or alternatively consisting of: 50% formamide, 5× SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing in 0.1× SSC at about 65°C. Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

[0076] “Near” or “adjacent,” as used herein is meant to include about 15residues on either side of the HIV-1 Gag CA-SP1 cleavage site; morepreferably about 10 residues on either side of the HIV-1 Gag CA-SP1cleavage site; and most preferably about 5 residues on either side ofthe HIV-1 Gag CA-SP1 cleavage site.

[0077] “Significantly,” where not otherwise defined herein, means +/−that observed or measured compared to the process or processing thatwould occur in the absence of the compound.

[0078] The invention also includes a virus comprising thepolynucleotides of the invention, and wherein the virus includes aretrovirus comprising said polynucleotides, and wherein the retrovirusmay be a member of the group consisting of HIV-1, HIV-2, HTLV-I,HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus(EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus(FIV), or feline leukemia virus (FeLV).

[0079] The invention further includes a polypeptide containing amutation in the CA-SP1 protein, said mutation which results in thedecrease in inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl) betulinic acid, and also wherein saidmutation is optionally located near the CA-SP1 cleavage site or locatedin the SP1 region of SEQ ID NO: 5 or SEQ ID NO: 7 (parentalpolynucleotide sequences) encoding the CA-SP1 protein. Said polypeptidemay be encoded by a polynucleotide selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, or maycomprise a sequence that is selected from the group consisting ofKARVLVEAMS (SEQ ID NO: 2) or KARVIAEVMS (SEQ ID NO: 3). The polypeptideof this invention may further be encoded by a polynucleotide whichhybridizes under highly stringent conditions to a polynucleotideselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8 or SEQ ID NO: 9. The invention also includes a polypeptide encodedby a polynucleotide which hybridizes to SEQ NO: 5, SEQ ID NO: 7 or SEQID NO: 10, which contains a mutation that results in decrease ininhibition of processing of p25 to p24 by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid, and also wherein said mutation is optionally located inthe SP1 region of CA-SP1. The polypeptide of this invention furtherincludes polypeptides that are part of a chimeric or fusion protein.Said chimeric proteins may be derived from species which include, butare not limited to: primates, including simian and human; rodentia,including rat and mouse; feline; bovine; ovine; including goat andsheep; canine; or porcine. Fusion proteins may include synthetic peptidesequences, bifunctional antibodies, peptides linked with proteins fromthe above species, or with linker peptides. Polypeptides of theinvention may be further linked with detectable labels; metal compounds;cofactors; chromatography separation tags, such as, but not limited to:histidine, protein A, or the like, or linkers; blood stabilizationmoieties such as, but not limited to: transferrin, or the like;therapeutic agents, and so forth.

[0080] The invention also includes an antibody which selectively bindsan amino acid sequence containing a mutation in the CA-SP1 protein thatresults in a decrease in the inhibition of processing of p25 (CA-SP1) top24 (CA) by 3-O-(3′,3′-dimethylsuccinyl) betulinic acid and also whereinsaid mutation is optionally located in the SP1 region of CA-SP1. Theinvention also includes an antibody which selectively binds thepolypeptide having a mutation which comprises a sequence that is one ofKARVLVEAMS (SEQ ID NO: 2), KARVIAEVMS (SEQ ID NO: 3). Said antibody canselectively bind the polypeptide encoded by a polynucleotide sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8 or SEQ ID NO: 9. Said antibody can also selectively bind thepolypeptide encoded by a polynucleotide which hybridizes under highlystringent conditions to a polynucleotide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9.The invention also includes an antibody that is selectively binds toSP1, which would enable one to distinguish SP1 from CA-SP1. Theinvention also includes an antibody that selectively binds CA, whichwould enable one to distinguish CA from CA-SP1. The inventionadditionally includes an antibody that selectively binds at or near theCA-SP1 cleavage site. The antibody of this invention may be a polyclonalantibody, a monoclonal antibody or said antibody may be chimeric orbifunctional, or part of a fusion protein. The invention furtherincludes a portion of any antibody of this invention, including singlechain, light chain, heavy chain, CDR, F(ab′)₂, Fab, Fab′, Fv, sFv, ordsFv, or any combinations thereof.

[0081] As used herein, an antibody “selectively binds” a target peptidewhen it binds the target peptide and does not significantly bind tounrelated proteins. The term “selectively binds” also comprisesdetermining whether the antibody selectively binds to the target mutantsequence relative to a native target sequence. An antibody which“selectively binds” a target peptide is equivalent to an antibody whichis “specific” to a target peptide, as used herein. An antibody is stillconsidered to selectively bind a peptide even if it also binds to otherproteins that are not substantially homologous with the target peptideso long as such proteins share homology with a fragment or domain of thepeptide target of the antibody. In this case, it would be understoodthat antibody binding to the peptide is still selective despite somedegree of cross-reactivity. In another embodiment, the determinationwhether the antibody selectively binds to the mutant target sequencecomprises: (a) determining the binding affinity of the antibody for themutant target sequence and for the native target sequences; and (b)comparing the binding affinities so determined, the presence of a higherbinding affinity for the mutant target sequence than for the nativeindicating that the antibody selectively binds to the mutant targetsequence.

[0082] The invention is further drawn to an antibody immobilized on aninsoluble carrier comprising any of the antibodies disclosed herein. Theantibody immobilized on an insoluble carrier includes multiple wellplates, culture plates, culture tubes, test tubes, beads, spheres,filters, electrophoresis material, microscope slides, membranes, oraffinity chromatography medium.

[0083] The invention also includes labeled antibodies, comprising adetectable signal. The labeled antibodies of this invention are labeledwith a detectable molecule, which includes an enzyme, a fluorescentsubstance, a chemiluminescent substance, horseradish peroxidase,alkaline phosphatase, biotin, avidin, an electron dense substance, and aradioisotope, or any combination thereof.

[0084] The invention further includes a method of producing a hybridomacomprising fusing a mammalian myeloma cell with a mammalian B cell thatproduces a monoclonal antibody which selectively binds an amino acidsequence containing a mutation in the CA-SP1 protein, said mutationresulting in a decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl) betulinic acid and a hybridoma producingany of the monoclonal antibodies disclosed herein. The invention furtherincludes a method of producing an antibody comprising growing ahybridoma producing the monoclonal antibodies disclosed herein in anappropriate medium and isolating the antibodies from the medium, as iswell known in the art. The invention also includes the production ofpolyclonal antibodies comprising the injection, either one injection ormultiple injections of any of the polypeptides of the inventions intoany animal known in the art to be useful for the production ofpolyclonal antibodies, including, but not limited to mouse, rat,hamster, rabbit, goat, sheep, deer, guinea pig, or primate, andrecovering the antibodies in sera produced therein. The inventionincludes high avidity or high affinity antibodies produced therein. Theinvention also includes B cells produced from the listed species to befurther used in cell fusion procedures for the manufacture of monoclonalantibody-producing hybridomas as disclosed herein.

[0085] The invention is further drawn to a kit comprising the antibodyor a portion thereof as disclosed herein, a container comprising saidantibody and instructions for use, a kit comprising the polypeptides ofthis invention and instructions for use and a kit comprising thepolynucleotide of the invention, a container comprising saidpolynucleotide and instructions for use, or any combinations thereof.These kits would include, but not be limited to nucleic acid detectionkits, which may, or may not, utilize PCR and immunoassay kits. Such kitsare useful for clinical diagnostic use and provide standardized reagentsas required in current clinical practice. These kits could eitherprovide information as to the presence or absence of mutations prior totreatment or monitor the progress of the patient during therapy. Thekits of the invention may also be used to provide standardized reagentsfor use in research laboratory studies.

[0086] Compounds useful in the present invention include, but are notlimited to those having the general Formula I and II:

[0087] I: Derivatives of Betulinic Acid (left) and Dihydrobetulinic Acid(right), or a pharmaceutically acceptable salt thereof, wherein,

[0088] R is a C₂-C₂₀ substituted or unsubstituted carboxyacyl,

[0089] R′ is hydrogen or a C₂-C₁₀ substituted and unsubstituted alkyl oraryl group. Preferred compounds are those wherein R is one of thesubstituents in Table 2 and R′ is hydrogen.

[0090] II: Derivatives of betulin and dihydrobetulin, or apharmaceutically acceptable salt thereof, wherein,

[0091] R₁ is a C₂-C₂₀ substituted or unsubstituted carboxyacyl,

[0092] R₂ is hydrogen or a C₂-C₂₀ substituted or unsubstitutedcarboxyacyl; and

[0093] R₃ is hydrogen, halogen, amino, optionally substituted mono- ordi-alkylamino, or —OR₄, where R₄ is hydrogen, C₁₋₄ alkanoyl, benzoyl, orC₂-C₂₀ substituted or unsubstituted carboxyacyl;

[0094] wherein the dashed line represents an optional double bondbetween C20 and C29.

[0095] Preferred compounds useful in the present invention are thosewhere R₁ is one of the substituents in Table 2, R₂ is hydrogen or one ofthe substituents in Table 1 2 and R₃ is hydrogen. TABLE 2 PreferredSubstituents

[0096] The most preferred compounds are 3-O-(3′,3′-dimethylsuccinyl)betulinic acid, 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid,3-O-(3′,3′-dimethylsuccinyl) betulin, and3-O-(3′,3′-dimethylsuccinylglutaryl) dihydrobetulin.

[0097] Compounds useful in the methods of the present invention includederivatives of betulinic acid and betulin that are presented in U.S.Pat. Nos. 5,679,828 and 6,172,110 respectively, and in U.S. applicationSer. Nos. 60/443,180 and 10/670,797, which are herein incorporated byreference. Additional useful compounds include oleanolic acidderivatives disclosed by Zhu et al. (Bioorg. Chem Lett. 11:3115-3118(2001)); oleanolic acid and promolic acid derivatives disclosed byKashiwada et al. (J. Nat. Prod. 61:1090-1095 (1998)); 3-O-acyl ursolicacid derivatives described by Kashiwada et al. (J. Nat. Prod.63:1619-1622 (2000)); and 3-alkylamido-3-deoxy-betulinic acidderivatives, disclosed by Kashiwada et al. (Chem. Pharm. Bull.48:1387-1390 (2000)). (All references incorporated by reference).

[0098] A particularly preferred compound is 3-O-(3′,3′-dimethylsuccinyl)betulinic acid.

[0099] Reaction of betulinic acid and dihydrobetulinic acid withdimethylsuccinic anhydride produced a mixture of3-O-(2′,2′-dimethylsuccinyl) and 3-O-(3′,3′-dimethylsuccinyl)-betulinicacid and dihydrobetulinic acid, respectively. The mixtures weresuccessfully separated by preparative scale HPLC yielding pure samples.The structures of these isomers were assigned by long-range ¹H-¹³C COSYexaminations.

[0100] The derivatives of betulinic acid and dihydrobetulinic acid ofthe present invention were all synthesized by refluxing a solution ofbetulinic acid or dihydrobetulinic acid, dimethylaminopyridine (1equivalent mol), and an appropriate anhydride (2.5-10 equivalent mol) inanhydrous pyridine (5-10 mL). The reaction mixture was then diluted withice water and extracted with CHCl₃. The organic layer was washed withwater, dried over MgSO₄, and concentrated under reduced pressure. Theresidue was chromatographed using silica gel colurnn orsemi-preparative-scale HPLC to yield the product.

[0101] Preparation of 3-O-(3′,3′-dimethylsuccinyl) betulinic acid: yield70% (starting with 542 mg of betulinic acid); crystallization from MeOHgave colorless needles; mp 274°-276° C.; [α]_(D) ¹⁹+23.5° (c=0.71),CHCl₃—MeOH [1:1]); Positive FABMS m/z 585 (M+H)⁺; Negative FABMS m/z 583(M−H)⁻; HR-FABMS calcd for C₃₆H₅₇O₆ 585.4155, found m/z 585.4161; ¹H NMR(pyridine-d₅): 0.73, 0.92, 0.97, 1.01, 1.05 (each 3H, s; 4-(CH₃)₂,8-CH₃, 10-CH₃, 14-CH₃), 1.55 (6H, s, 3′-CH₃×2), 1.80 (3H, s, 20-CH₃),2.89, 2.97 (each 1H, d, J=15.5 Hz, H-2′), 3.53 (1H, m, H-19), 4.76 (1H,dd, J=5.0, 11.5 Hz, H-3), 4.78, 4.95 (each 1H, br s, H-30).

[0102] 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid: yield 24.5%(starting with 155.9 mg of dihydrobetulinic acid); crystallization fromMeOH—H₂O gave colorless needles; mp 291°-292° C.; [α]_(D)²⁰−13.4°(c=1.1, CHCl₃—MeOH [1:1], ¹H NMR (pyridine-d₅): 0.85, 0.94 (each3H, d, J=7.0 Hz; 20-(CH₃)₂), 0.75, 0.93, 0.97, 1.01, 1.03 (each 3H, s;4-(CH₃)₂, 8-CH₃, 10-CH₃, 14-CH₃), 1.55 (6H, s; 3′-CH₃×2), 2.89, 2.97(each 1H, d, J=15.5 Hz; H-2′), 4.77 (1H, dd, J=5.0, 11.0 Hz, H-3); Anal.Calcd for C₃₆H₅₈O₆.5/2H₂O: C 68.43, H 10.04; found C 68.64, H 9.78.

[0103] The synthesis of 3-O-(3′,3′-dimethylglutaryl) betulinic acid wasdisclosed U.S. Pat. No. 5,679,828, as COMPOUND NO. 4.

[0104] 3-O-(3′,3′-dimethylglutaryl) dihydrobetulinic acid: yield 93.3%(starting with 100.5 mg of dihyrdobetulinic acid); crystallization fromneedles MeOH—H₂O gave colorless needles; mp 287°-289° C.; [α]_(D)²⁰−17.9° (c=0.5, CHCl₃—MeOH[1:1]); ¹H-NMR (pyridine-d₅): 0.86, 0.93(each 3H, d, J=6.5 Hz; 20-(CH₃)₂), 0.78, 0.92, 0.96, 1.02, 1.05 (each3H, s; 4-(CH₃)₂, 8-CH₃, 10-CH₃, 14-CH₃), 1.38, 1.39 (each 3H, s;3′-CH₃×2), 2.78 (4H, m, H₂-2′ and 4′), 4.76 (1H, dd, J=4.5, 11.5 Hz;H-3). Anal. Calcd for C₃₇H₆₀O₆ : C 73.96, H 10.06; found C 73.83, H10.10.

[0105] The synthesis for 3-O-diglycolyl-betulinic acid was disclosed inU.S. Pat. No. 5,679,828, as COMPOUND NO. 5.

[0106] 3-O-diglycolyl-dihydrobetulinic acid: yield 79.2% (starting with103.5 mg of dihydrobetulinic acid); an off-white amorphous powder;[α]_(D) ²⁰−9.8° (c=1.1, CHCl₃—MeOH[1:1]); ¹H-NMR (pyridine-d₅): 0.79,0.87 (each 3H, d, J=6.5 Hz; 20-(CH₃)₂), 0.87, 0.88, 0.91, 0.98, 1.01(each 3H, s; 4-(CH₃)₂, 8-CH₃, 10-CH₃, 14-CH₃), 4.21, 4.23 (each 2H, s,H₂-2′ and 4′), 4.57 (1H, dd, J=6.5, 10.0 Hz, H-3); Anal. Calcd forC₃₄H₅₄O₇.2H₂O: C 66.85, H 9.57; found C 67.21, H 9.33.

[0107] The syntheses of 3-O-(3′,3′-dimethylsuccinyl) betulin and3-O-(3′,3′-dimethylglutaryl) betulin were disclosed in U.S. application10/670,797.

[0108] The method of inhibiting an HIV-1 replication in cells of ananimal includes a compound of Formula I or Formula II, above, which is aderivative of betulinic acid, betulin, or dihydrobetulinic acid ordihydrobetulin and which includes the preferred substituents of Table 2.Preferred compounds include but are not limited to3-O-(3′,3′-dimethylsuccinyl) betulinic acid,3-O-(3′,3′-dimethylsuccinyl) betulin, 3-O-(3′,3′-dimethylglutaryl)betulin, 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl) betulinic acid, (3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, and3-O-diglycolyl-dihydrobetulinic acid.

[0109] The method disclosed herein, further comprises contacting saidcells with one or more drugs selected from the group consisting ofanti-viral agents, anti-fungal agents, anti-bacterial agents,anti-cancer agents, immunostimulating agents, and combinations thereof.The method may include the treatment of human blood products.

[0110] The invention may also be used in conjunction with a method oftreating cancer comprising the administration to an animal of one ormore anti-neoplastic agents, exposing an animal to a cancer cell-killingamount of radiation, or a combination of both.

[0111] The invention further includes a method for identifying compoundsthat inhibit HIV-1 replication in cells of an animal disclosed herein,said method comprising:

[0112] a. contacting a Gag protein comprising a CA-SP1 cleavage sitewith a test compound;

[0113] b. adding a labeled substance that selectively binds at or nearthe CA-SP1 cleavage site; and

[0114] c. measuring the binding of the test compound at or near theCA-SP1 cleavage site.

[0115] Labeled substances or molecules include labeled antibodies orlabeled DSB and the label includes an enzyme, fluorescent substance,chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, such as gold,osmium tetroxide, lead or uranyl acetate, and radioisotope, antibodieslabeled with such substances of molecules or a combination thereof. Theassays could include, but are not limited to ELISA, single and doublesandwich techniques, immunodiffusion or immunoprecipitation techniques,as known in the art (“Immunoassay Handbook, 2^(nd) ed.,” D. Wild, NaturePublishing Group, (2001)). Said methods of identifying also couldinclude, but are not limited to Western blot assays, calorimetricassays, light and electron microscopic techniques, confocal microscopy,or other techniques known in the art.

[0116] A method of identifying compounds that inhibit HIV replication incells of an animal further comprises:

[0117] a. contacting a Gag protein comprising a wild-type CA-SP1cleavage site, with HIV-1 protease in the presence of a test compound;

[0118] b. separately, contacting a Gag protein comprising a mutantCA-SP1 cleavage site or a protein comprising an alternative proteasecleavage site with HIV-1 protease in the presence of the test compound;and

[0119] c. comparing the cleavage of the native wild-type Gag protein tothe amount of cleavage of the mutant Gag protein or to the amount ofcleavage of the peptide comprising an alternative protease cleavagesite.

[0120] Step (b) above is performed as a control in order to eliminatecompounds that might bind directly to, and therefore inhibit, theprotease enzyme. The above method also includes the method wherein thewild-type CA-SP1, mutant CA-SP1 or alternative protease cleavage site iscontained within a polypeptide fragment or recombinant peptide.

[0121] The method for identifying compounds that inhibit HIV-1 disclosedherein also, includes a method wherein said peptide or protein islabeled with a fluorescent moiety and a fluorescence quenching moiety,each bound to opposite sides of the CA-SP1 cleavage site, and whereinsaid detecting comprises measuring the signal from the fluorescentmoiety, or wherein said peptide or protein is labeled with twofluorescent moieties, each bound to opposite sides of the CA-SP1cleavage site, and wherein said detecting comprises measuring thetransfer of fluorescent energy from one moiety to the other in thepresence of the test compound and HIV-1 protease and comparing saidtransfer of fluorescent energy to that observed when the same procedureis applied to a peptide that comprises a sequence containing a mutationin the CA-SP1 cleavage site or that comprises a sequence containinganother cleavage site. Examples of fluorescence-based assays of proteaseactivity are well known in the art. In one such example, a proteasesubstrate is labeled with green fluorescent dye molecules, whichfluoresce when the substrate is cleaved by the protease enzyme(Molecular Probes, Protease Assay Kit).

[0122] The method of comparing the cleavage, above, also includes usinga labeled antibody that selectively binds CA or SP1 in order to measurethe extent to which the test compound inhibits CA-SP1 cleavage. Theantibody can be labeled with a molecule selected from the groupconsisting of enzyme, fluorescent substance, chemiluminescent substance,horseradish peroxidase, alkaline phosphatase, biotin, avidin, electrondense substance, and radioisotope, or combinations thereof. The methodalso includes the use of an antibody to a specific epitope tag sequenceto selectively detect p25 or SP1 into which the amino acid sequence forthat epitope tag has been engineered according to standard methods inthe art. As an example, the sequence of the FLAG epitope tag(Sigma-Aldrich) could be inserted into the p2 (SP1) region of Gag byoligonucleotide-directed mutagenesis of a Gag expression plasmid. Thepresence of the SP1 region in the cell-expressed protein could then bedetected using commercially available anti-FLAG monoclonal antibodies(Sigma-Aldrich). (Hopp, T. P. Biotechnology 6: 1204-1210 (1988)).

[0123] The method also includes the addition of a compound to cellsinfected with HIV-1 and the detection of CA-SP1 cleavage products bylysing and analyzing the cells or the released virions. The methodincluded in the invention can be performed using a western blot analysisof viral proteins and detecting p25 using an antibody that selectivelybinds p25 or wherein said mixture is analyzed by performing a gelelectrophoresis of viral proteins and imaging of metabolically labeledproteins, or wherein the mixture is analyzed using immunoassays that usean antibody that selectively binds p25 or selectively binds SP1 todistinguish p25 from p24. For example, a microwell assay can beperformed where p25 in detergent-solubilized virus is captured using anantibody selectively binds SP1 that is bound to the plastic multiplewell plate. Following a washing step, bound p25 is detected using anantibody to p24 that is conjugated to an appropriate detection reagent(e.g. alkaline phosphatase for an enzyme-linked immunosorbent assay).Virus released by cells treated with compounds that act via thismechanism will have increased levels of p25 compared with untreatedvirions.

[0124] The disclosed method is drawn to an antibody that selectivelybinds p25, or an antibody that selectively binds SP1, which is labeledwith a molecule selected from the group consisting of enzyme,fluorescent substance, chemiluminescent substance, horseradishperoxidase, alkaline phosphatase, biotin, avidin, electron densesubstance, and radioisotope, or combinations thereof. The invention alsoincludes the use of an antibody to a specific epitope tag sequence toselectively detect p25 or SP1 into which the amino acid sequence forthat epitope tag has been engineered according to standard methods inthe art.

[0125] “Infected cells,” as used herein, includes cells infectednaturally by membrane fusion and subsequent insertion of the viralgenome into the cells, or transfection of the cells with viral geneticmaterial through artificial means. These methods include, but are notlimited to, calcium phosphate transfection, DEAE-dextran mediatedtransfection, microinjection, lipid-mediated transfection,electroporation or infection.

[0126] The invention may be practiced by infecting target cells in vitrowith an infectious strain of HIV and in the presence of test compound,under appropriate culture conditions and for varying periods of time.Infected cells or supernatant fluid can be processed and loaded onto apolyacrylamide gel for the detection of virus levels, by methods thatare well known in the art. Non-infected and non-treated cells can beused as negative and positive infection controls, respectively.Alternatively, the invention may be practiced by culturing the targetcells in the presence of test compound prior to infecting the cells withan HIV strain.

[0127] The invention also includes a method for identifying compoundsthat inhibit HIV-1 replication in the cells of an animal, comprising:

[0128] a. contacting a test compound with wild-type virus isolates andseparately with virus isolates resistant to 3-O-(3′,3′-dimethylsuccinyl)betulinic acid; and

[0129] b. selecting test compounds that are more active against thewild-type virus isolate compared with virus isolates that are resistantto 3-O-(3′,3′-dimethylsuccinyl) betulinic acid.

[0130] This invention further includes a method for identifyingcompounds that act by any of the abovementioned mechanism, involvingtreating HIV-1 infected or transfected cells with a compound thenanalyzing the virus particles released by compound-treated cells bythin-sectioning and transmission electron microscopy, by standardmethods well known in the art. A compound acts by the abovementionedmechanism if particles are detected that exhibit spherical condensedcores that are acentric with respect to the viral particle and acrescent-shaped electron-dense layer just inside the viral membrane.

[0131] For electron microscopic studies, infected cells or centrifugedvirus pellets obtained from the supernatant fluid can be contacted witha fixative, such as glutaraldehyde or freshly-made paraformaldehyde,and/or osmium tetroxide or other electron microscopy compatible fixativethat is known in the art. The virus from the supernatant fluid or thecells, is dehydrated and embedded in an electron-lucent polymer such asan epoxy resin or methacrylate, thin sectioned using an ultramicrotome,stained using electron dense stains such as uranyl acetate, and/or leadcitrate, and viewed in a transmission electron microscope. Non-infectedand non-treated cells can be used as negative and positive infectioncontrols, respectively. Alternatively, the invention may be practiced byculturing the target cells in the presence of test compound prior toinfecting the cells with an HIV strain. Maturation defects caused by thecompounds of the present invention are determined by the presence ofmorphologically aberrant viral particles, compared with controls, asdescribed herein.

[0132] For cell culture studies, the virus-infected cells may beobserved for the formation of syncytia, or the supernatant may be testedfor the presence of HIV particles. Virus present in the supernatant maybe harvested to infect other naive cultures to determine infectivity.

[0133] Also included in the invention, is a method of determining if anindividual is infected with HIV-1, is susceptible to treatment by acompound that inhibits p25 processing, the method involves taking bloodfrom the patient, genotyping the viral RNA and determining whether theviral RNA contains mutations in the CA-SP1 cleavage site.

[0134] The invention also includes a method for identifying compoundsthat act by the abovementioned mechanisms, involving testing by acombination of the methods disclosed herein.

[0135] HIV Gag protein and fragments thereof for use in theaforementioned assays may be expressed or synthesized using a variety ofmethods familiar to those skilled in the art. Gag can be produced in anin vitro transcription and translation system using a rabbitreticulocyte lysate. Gag expressed in this system has been shown to beprocessed sequentially in a pattern similar to that observed in infectedcells (Pettit, S. C. et al. J. Virol. 76:10226-10233 (2002)). Moreover,Gag expressed by this method is capable of assembling into immatureviral particles when fused to a heterologous type D retroviralcytoplasmic self-assembly domain (Sakalian, M. et al., J. Virol.76:10811-10820 (2002)). The plasmid pDAB72, available from the NIH AIDSReagent Program can be used for this purpose (Erickson-Viitanen, S. etal., AIDS Res. Hum. Retroviruses. 5:577-91 (1989); Sidhu M. K. et al.,Biotechniques, 18:20, 22, 24 (1995)). Other in vitrotranscription/translation systems based on wheat germ or bacteriallysates can also be used for this purpose. HIV Gag may also be expressedin transfected cells using a variety of commercially availableexpression vectors. The plasmid p55-GAG/GFP, available from the NIH AIDSReagent Program, may be used to express an HIV Gag-green fluorescentprotein fusion protein in mammalian cells for drug interaction studies(Sandefur, S. et al., J. Virol. 72:2723-2732 (1998)). This constructwould permit the capture and purification of Gag fusion protein usingGFP-specific monoclonal antibodies. In addition, Gag may be expressed incells using recombinant viral vectors, such as those used in thevaccinia virus, adenovirus, or baculovirus systems. Gag can alsoexpressed by infecting cells with HIV or by transfecting cells withproviral DNA. Finally, Gag may be expressed in yeast or bacterial cellstransformed with the appropriate expression vectors.

[0136] In addition to Gag proteins expressed in cells or in vitro usingcell lysates, peptides corresponding to various regions of Gag may becommercially synthesized from using standard peptide synthesistechniques.

[0137] The invention further encompasses compounds identified by themethod of this invention and/or a compound which inhibits HIV-1replication according to the methods of this invention andpharmaceutical compositions comprising one or more compounds asdisclosed herein, or pharmaceutically acceptable salts, esters orprodrugs thereof, and pharmaceutically acceptable carriers.

[0138] Also included in the invention are compounds that are useful inthe present invention, which include compounds of Formula I and FormulaII, above. Preferred compounds include 3-O-(3′,3′-dimethylsuccinyl)betulinic acid, 3-O-(3′,3′-dimethylsuccinyl) betulin,3-O-(3′,3′-dimethylglutaryl) betulin, 3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid, 3-O-(3′,3′-dimethylglutaryl) betulinic acid,(3′,3′-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, 3-O-diglycolyl-dihydrobetulinic acid, and any combination thereof.

[0139] Also, included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free acid form with a suitable organic orinorganic base and isolating the salt thus formed. These may includecations based on the alkali and alkali earth metals, such as sodium,lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium and amine cations including, butnot limited to ammonium, tetra-methylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, ethylamine,N-methyl-glucamine and the like.

[0140] Compounds of Formulas I and II according to the present inventionhave been found to possess anti-retroviral, particularly anti-HIV,activity. The salts and other formulations of the present invention areexpected to have improved water solubility, and enhanced oralbioavailability. Also, due to the improved water solubility, it will beeasier to formulate the salts of the present invention intopharmaceutical preparations. Further, compounds of Formula I and IIaccording to the present invention are expected to have improvedbiodistribution properties.

[0141] This invention also includes a pharmaceutical compositioncomprising a compound that inhibits processing of the viral Gag p25protein (CA-SP1) to p24 (CA), but has no significant effect on other Gagprocessing steps, or that inhibits the maturation of virus particlesreleased from treated infected cells, such as the compounds of Formula Iand II. The invention includes a pharmaceutical composition comprisingone or more compounds disclosed herein, or pharmaceutically acceptablesalts, esters or prodrugs thereof, and pharmaceutically acceptablecarriers, wherein said compound is of Formula I or II above, orpreferably, wherein said compound is selected from the group consistingof 3-O-(3′,3′-dimethylsuccinyl) betulinic acid,3-O-(3′,3′-dimethylsuccinyl) betulin, 3-O-(3′,3′-dimethylglutaryl)betulin, 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl) betulinic acid, (3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, and3-O-diglycolyl-dihydrobetulinic acid. The pharmaceutical compositionsaccording to the invention, further comprise one or more drugs selectedfrom an anti-viral agent, anti-fungal agent, anti-cancer agent or animmunostimulating agent.

[0142] Pharmaceutical compositions of the present invention can compriseat least one of the compounds of Formula I or II disclosed herein.Pharmaceutical compositions according to the present invention can alsofurther comprise other anti-viral agents such as, but not limited to,AZT (zidovudine, RETROVIR®, Glaxo Wellcome), 3TC (lamivudine, COMBIVIR®,Glaxo Wellcome), ddI (didanosine, VIDEX®, Bristol-Myers Squibb), ddC(zalcitabine, HIVID®, Hoffmann-La Roche), D4T (stavudine, ZERIT®,Bristol-Myers Squibb), abacavir (ZIAGEN®, Glaxo Wellcome), nevirapine(VIRAMUNE®, Boehringher Ingelheim), delavirdine (Pharmacia and Upjohn),efavirenz (SUSTIVA®, DuPont Pharmaceuticals), saquinavir (INVIRASE®,FORTOVASE®, Hoffmann-La Roche), ritonavir (NORVIR®, AbbottLaboratories), indinavir (CRIXIVAN®, Merck and Company), nelfinavir(VIRACEPT®, Agouron Pharmaceuticals), amprenavir (AGENERASE®, GlaxoWellcome), adefovir (PREVEON®, HEPSERA®, Gilead Sciences), atazanavir(Bristol-Myers Squibb), and hydroxyurea (HYDREA®, Bristol-MeyersSquibb), or any other antiretroviral drugs or antibodies in combinationwith each other, or associated with a biologically based therapeutic,such as, for example, gp41-derived peptides enfuvirtide (FUZEON®, Rocheand Trimeris) and T-1249, or soluble CD4, antibodies to CD4, andconjugates of CD4 or anti-CD4, or as additionally presented herein.

[0143] Additional suitable antiviral agents for optimal use with one ofthe compounds of Formula I or II of the present invention can include,but are not limited to, AL-721 (lipid mixture) manufactured by EthigenCorporation and Matrix Research Laboratories; amphotericin B(FUNGIZONE®; Ampligen (mismatched RNA) developed by DuPont/HEM Research;anti-AIDS antibody (Nisshon Food); 1 AS-101 (heavy metal basedimmunostimulant); BETASERON® (β-interferon, Triton Biosciences);butylated hydroxytoluene; Carrosyn (polymannoacetate); Castanospermine;Contracan (stearic acid derivative); Creme Pharmatex (containingbenzalkonium chloride) manufactured by Pharmalec; CS-87 (5-unsubstitutedderivative of zidovudine); penciclovir (DENAVIR® Novartis); famciclovir(FAMVIR® Novartis); acyclovir (ZOVIRAX® Glaxo Wellcome); HPMPC(cytofovir, VISTIDE® Gilead); DHPG, (ganciclovir, CYTOVENE®, RochePharmaceuticals); dextran sulfate; D-penicillamine (3-mercapto-D-valine)manufactured by Carter-Wallace and Degussa Pharmaceutical; FOSCARNET®(trisodium phosphonoformate; Astra AB); fusidic acid manufactured by LeoLovens; glycyrrhizin (a constituent of licorice root); HPA-23(ammonium-21-tungsto-9-antimonate; Rhone-Poulenc Sante); human immunevirus antiviral developed by Porton Products International; ORNIDYL®(eflornithine; Merrell-Dow); nonoxynol; pentamidine isethionate(PENTAM-300) manufactured by Lypho Med; Peptide T (octapeptide sequence)manufactured by Peninsula Laboratories; Phenytoin (Warner-Lambert); INHor isoniazid; ribavirin (RIFADIN®, Aventis); (VIRAZOLE®, ICNPharmaceuticals); rifabutin, ansamycin (MYCOBUTIN® Pfizer); CD4-IgG2(Progenics Pharmaceuticals) or other CD4-containing or CD4-basedmolecules; Trimetrexate manufactured by Warner-Lambert Company; SK-818(germanium-derived antiviral) manufactured by Sanwa Kagaku; suramin andanalogues thereof manufactured by Miles Pharmaceuticals; UA001manufactured by Ueno Fine Chemicals Industry; and WELLFERON®(α-interferon, Glaxo Wellcome).

[0144] Pharmaceutical compositions of the present invention can alsofurther comprise immunomodulators. Suitable immunomodulators foroptional use with a betulinic acid or betulin derivative of the presentinvention in accordance with the present invention can include, but arenot limited to: ABPP (Bropririmine); Ampligen (mismatched RNA)DuPont/HEM Research; anti-human interferon-α-antibody (AdvanceBiotherapy and Concepts); anti-AIDS antibody (Nisshon Food); AS-101(heavy metal based immunostimulant; ascorbic acid and derivativesthereof; interferon-β; Ciamexon (Boehringer-Mannheim); cyclosporin;cimetidine; CL-246,738 (American Cyanamid); colony stimulating factors,including GM-CSF (Sandoz, Genetics Institute); dinitrochlorobenzene;HE2000 (Hollis-Eden Pharmaceuticals); inteferon-γ; glucan; hyperimmunegamma-globulin (Bayer); IMREG-1 (leukocyte dialyzate) and IMREG-2 (IMREGCorp.); immuthiol (sodium diethylthiocarbamate) (Institut Merieux);interleukin-1 (Cetus Corporation, Hoffmann-LaRoche; Immunex),interleukin-2 (IL-2) (Chiron Corporation), isoprinosine (inosinepranobex), Krestin (Sankyo), LC-9018 (Yakult), lentinan(Ajinomoto/Yamanouchi); LF-1695 (Fournier), methionine-enkephalin (TNIPharmaceuticals; Sigma Chemicals), Minophagen C; muramyl tripeptide,MTP-PE (Ciba-Geigy), naltrexone (TREXAN® DuPont); Neutropin, RNAimmunomodulator (Nippon Shingaku), REMUNE® (Immune ResponseCorporation), RETICULOSE® (Advanced Viral Research Corporation),shosaikoto, ginseng, thymic humoral factor, TP-05 (Thymopentin, OrthoPharmaceuticals), thymosin factor 5, thymosin 1 (ZYDAXIN®, SciClone),thymostimulin, TNF (tumor necrosis factor Genentech), and vitaminpreparations.

[0145] Pharmaceutical compositions of the present invention can alsofurther comprise anti-cancer therapeutic agents. Suitable anti-cancertherapeutic agents for optional use include an anti-cancer compositioneffective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said anti-cancer agent,which can be used for combination therapy include, but are not limitedto alkylating agents, such as busulfan, cis-platin, mitomycin C, andcarboplatin antimitotic agents, such as colchicine, vinblastine, taxols,such as paclitaxel (TAXOL®, Bristol-Meyers Squibb) docetaxel (TAXOTERE®,Aventis), topo I inhibitors, such as camptothecin, irinotecan andtopotecan (HYCAMTIN®, SmithKline Beecham), topo II inhibitors, such asdoxorubicin, daunorubicin and etoposides such as VP16; RNA/DNAantimetabolites, such as 5-azacytidine, 5-fluorouracil and methotrexate,DNA antimetabolites, such as 5-fluoro-2′-deoxy-uridine, ara-C,hydroxyurea, thioguanine, and antibodies, such as trastuzumab(HERCEPTIN®, Genentech), and rituximab (RITUXAN®, Genentech and IdecPharmaceuticals), melphalan, chlorambucil, cyclophosamide, ifosfamide,vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin,mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid,tamoxifen, alanosine, and combinations thereof.

[0146] The invention further provides methods for providinganti-bacterial therapeutics, anti-parasitic therapeutics, andanti-fungal therapeutics, for use in combination with the compounds ofthe invention and pharmaceutically-acceptable salts thereof. Examples ofanti-bacterial therapeutics include compounds such as penicillins,ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin,cephalexin, cepharadine, cefadoxil, cefaclor, cefoxitin, cefotaxime,ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, imipenem,clavulanate, timentin, sulbactam, erythromycin, neomycin, gentamycin,streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin,quinolones, rifampin, sulfonamides, bacitracin, polymyxin B, vancomycin,doxycycline, methacycline, minocycline, tetracycline, amphotericin B,cycloserine, ciprofloxacin, norfloxacin, isoniazid, ethambutol, andnalidixic acid, as well as derivatives and altered forms of each ofthese compounds.

[0147] Examples of anti-parasitic therapeutics include bithionol,diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine,niridazole, oxamniquine and other quinine derivatives, piperazinecitrate, praziquantel, pyrantel pamoate and thiabendazole, as well asderivatives and altered forms of each of these compounds.

[0148] Examples of anti-fungal therapeutics include amphotericin B,clotrimazole, econazole nitrate, flucytosine, griseofulvin, ketoconazoleand miconazole, as well as derivatives and altered forms of each ofthese compounds. Anti-fungal compounds also include aculeacin A andpapulocandin B.

[0149] The term “prodrug”, as used herein refers to compounds whichundergo biotransformation prior to exhibiting their pharmacologicaleffects. The chemical modification of drugs to overcome pharmaceuticalproblems has also been termed “drug latentiation.” Drug latentiation isthe chemical modification of a biologically active compound to form anew compound which upon in vivo enzymatic attack will liberate theparent compound. The chemical alterations of the parent compound aresuch that the change in physicochemical properties will affect theabsorption, distribution and enzymatic metabolism. The definition ofdrug latentiation has also been extended to include nonenzymaticregeneration of the parent compound. Regeneration takes place as aconsequence of hydrolytic, dissociative, and other reactions notnecessarily enzyme mediated. The terms “prodrugs,” “latentiated drugs,”and “bioreversible derivatives” are used interchangeably. By inference,latentiation implies a time lag element or time component involved inregenerating the bioactive parent molecule in vivo. The term “prodrug”is general in that it includes latentiated drug derivatives as well asthose substances which are converted after administration to the actualsubstance. The term “prodrug” is a generic term for agents which undergobiotransformation prior to exhibiting their pharmacological actions.

[0150] The preferred animal subject of the present invention is amammal. By the term “mammal” is meant an individual belonging to theclass Mammalia. The invention is particularly useful in the treatment ofhuman patients.

[0151] The term “treating” means the administering to subjects acompound of Formula I or II or a compound identified by one or moreassays within the present invention, for purposes which can includeprevention, amelioration, or cure of a retroviral-related pathology.Said compounds for treating a subject that are identified by one or moreassays within the present inventions are identified as compounds whichhave the ability to disrupt Gag processing, described herein.

[0152] The term “inhibits the interaction” as used herein, meanspreventing, or reducing the rate of, direct or indirect association ofone or more molecules, peptides, proteins, enzymes, or receptors; orpreventing or reducing the normal activity of one or more molecules,peptides, proteins, enzymes or receptors.

[0153] Medicaments are considered to be provided “in combination” withone another if they are provided to the patient concurrently or if thetime between the administration of each medicament is such as to permitan overlap of biological activity.

[0154] In one preferred embodiment, at least one compound of Formula Ior II above comprises a single pharmaceutical composition.

[0155] Pharmaceutical compositions for administration according to thepresent invention can comprise at least one compound of Formula I or IIabove or compounds identified by one or more assays within the presentinvention. Said compounds for treating a subject that are identified byone or more assays within the present inventions are identified ascompounds which have the ability to disrupt Gag processing, describedherein. The compounds according to the present invention are furtherincluded in a pharmaceutically acceptable form optionally combined witha pharmaceutically acceptable carrier. These compositions can beadministered by any means that achieve their intended purposes. Amountsand regimens for the administration of a compound of Formula I or IIaccording to the present invention can be determined readily by thosewith ordinary skill in the clinical art of treating a retroviralpathology.

[0156] For example, administration can be by parenteral, such assubcutaneous, intravenous, intramuscular, intraperitoneal, transdermal,transmucosal, ocular, rectal, intravaginal, or buccal routes.Alternatively, or concurrently, administration can be by the oral route.The administration may be as an oral or nasal spray, or topically, suchas powders, ointments, drops or a patch. The dosage administered dependsupon the age, health and weight of the recipient, type of previous orconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

[0157] Compositions within the scope of this invention include allcompositions comprising at least one compound of Formula I or II aboveaccording to the present invention in an amount effective to achieve itsintended purpose. While individual needs vary, determination of optimalranges of effective amounts of each component is within the skill of theart. Typical dosages comprise about 0.1 to about 100 mg/kg body weight.The preferred dosages comprise about 1 to about 100 mg/kg body weight ofthe active ingredient. The most preferred dosages comprise about 5 toabout 50 mg/kg body weight.

[0158] Administration of a compound of the present invention can alsooptionally include previous, concurrent, subsequent or adjunctivetherapy using immune system boosters or immunomodulators. In addition tothe pharmacologically active compounds, a pharmaceutical composition ofthe present invention can also contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations, particularlythose preparations which can be administered orally and which can beused for the preferred type of administration, such as tablets, dragees,and capsules, and also preparations which can be administered rectally,such as suppositories, as well as suitable solutions for administrationby injection or orally, contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound(s), togetherwith the excipient.

[0159] Pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

[0160] Suitable excipients are, e.g., fillers such as saccharide, forexample, lactose or sucrose, mannitol or sorbitol; cellulosepreparations and/or calcium phosphates, such as tricalcium phosphate orcalcium hydrogen phosphate; as well as binders such as starch paste,using, for example, maize starch, wheat starch, rice starch, potatostarch, gelatin, tragacanth, cellulose, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents can be addedsuch as the above-mentioned starches and also carboxymethyl starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are, above all,flow-regulating agents and lubricants, for example, silica, talc,stearic acid or salts thereof, such as magnesium stearate or calciumstearate, and/or polyethylene glycol. Dragee cores are provided withsuitable coatings which, if desired, are resistant to gastric juices.For this purpose, concentrated saccharide solutions can be used, whichcan optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigments can be added to thetablets or dragee coatings, for example, for identification or in orderto characterize combinations of active compound doses.

[0161] Other pharmaceutical preparations which an be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichcan be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils or liquidparaffin. In addition, stabilizers can be added.

[0162] Possible pharmaceutical preparations which can be used rectallyinclude, for example, suppositories which consist of a combination ofthe active compounds with a suppository base. Suitable suppository basesare, for example, natural or synthetic triglycerides, or paraffinhydrocarbons. In addition, it is also possible to use gelatin rectalcapsules which consist of a combination of the active compounds with abase. Possible base materials include, for example, liquidtriglycerides, polyethylene glycols, or paraffin hydrocarbons.

[0163] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions can be administered.Suitable lipophilic solvents or vehicles include fatty oils, such assesame oil, or synthetic fatty acid esters, such as ethyl oleate,triglycerides or glycol-400. Aqueous injection suspensions that cancontain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension can also contain stabilizers.

[0164] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide, oils such as cottonseed,groundnut, corn, germ, olive, castor, and sesame oils, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

[0165] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, cellulose,microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, and combinations thereof.

[0166] Pharmaceutical compositions for topical administration includeformulations appropriate for administration to the skin, mucosa,surfaces of the lung or eye. Compositions may be prepared as apressurized or non-pressurized dry powder, liquid or suspension. Theactive ingredients in non-pressurized powdered formulations may beadmixed in a finely divided form in a pharmaceutically-acceptable inertcarrier, including but not limited to mannitol, fructose, dextrose,sucrose, lactose, saccharin or other sugars or sweeteners.

[0167] The pressurized composition may contain a compressed gas, such asnitrogen, or a liquefied gas propellant. The propellant may also containa surface-active ingredient, which may be a liquid or solid non-ionic oranionic agent. The anionic agent may be in the form of a sodium salt.

[0168] A formulation for use in the eye would comprise apharmaceutically acceptable ophthalmic carrier, such as an ointment,oils, such as vegetable oils, or an encapsulating material. The regionsof the eye to be treated include the corneal region, or internal regionssuch as the iris, lens, ciliary body, anterior chamber, posteriorchamber, aqueous humor, vitreous humor, choroid or retina.

[0169] Compositions for rectal administration may be in the form ofsuppositories. Compositions for use in the vagina may be in the form ofsuppositories, creams, foams, or in-dwelling vaginal inserts.

[0170] The compositions may be administered in the form of liposomes.Liposomes may be made from phospholipids, phosphatidyl cholines(lecithins) or other lipoidal compounds, natural or synthetic, as knownin the art. Any non-toxic, pharmacologically acceptable lipid capable offorming liposomes may be used. The liposomes may be multilamellar ormono-lamellar.

[0171] A pharmaceutical formulation for systemic administrationaccording to the invention can be formulated for enteral, parenteral ortopical administration. Indeed, all three types of formulation can beused simultaneously to achieve systemic administration of the activeingredient.

[0172] Suitable formulations for oral administration include hard orsoft gelatin capsules, dragees, pills, tablets, including coatedtablets, elixirs, suspensions, syrups or inhalations and controlledrelease forms thereof.

[0173] The compounds of Formula I or II above or compounds identified byone or more assays within the present invention and have the ability todisrupt Gag processing, can also be administered in the form of animplant when compounded with a biodegradable slow-release carrier.Alternatively, the compounds of the present invention can be formulatedas a transdermal patch for continuous release of the active ingredient.

[0174] The following examples are illustrative only and are not intendedto limit the scope of the invention as defined by the appended claims.It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods of the presentinvention without departing from the spirit and scope of the invention.Thus, it is intended that the present invention covers the modificationsand variations of this invention provided they come within the scope ofthe appended claims and their equivalents.

EXAMPLES Example 1

[0175] Anti-Viral Activity Against Primary HIV-1 Isolates:

[0176] A robust virus inhibition assay was used to evaluate theanti-viral activity of DSB against primary HIV-1 isolates propagated inPMBC. Briefly, serial dilutions of DSB were made in medium into 96-welltissue culture plates. 25-250 TCID₅₀ of virus and 5×10⁵ PHA-stimulatedPBMCs were added to each well. On days 1, 3 and 5 post-infection, mediawas removed from each well and replaced with fresh media containing DSBat the appropriate concentration. On day 7 post-infection, culturesupernatant was removed from each well for p24 detection of virusreplication and 50% inhibitory concentrations (IC₅₀) were calculated bystandard methods.

[0177] Table 3 shows the potent anti-viral activity of DSB against apanel of primary HIV-1 isolates. DSB exhibits levels of activity similarto approved drugs that were tested in parallel. Importantly, theactivity of DSB was not restricted by co-receptor usage. TABLE 3 Table3: Inhibitory activity (IC₅₀) of DSB and two approved drugs against apanel of primary Clade B HIV-1 isolates. Clinical HIV-1 isolates denotedby * were isolated at Panacos. All other virus isolates were obtainedfrom the NIH AIDS Reference Repository. Co-Receptor IC₅₀ (nM) VirusIsolate # usage DSB AZT Nevirapine BZ167 X4 4.0 2.2 31.2 92HT599 X4 9.85.8 25.3 US1 R5 5.6 0.9 22.1 19101N* R5 3.8 2.4 59.4 3401N* R5/X4 12.017.5 32.1 92US723 R5/X4 4.6 1.2 26.8 22101N* R5/X4 2.6 0.9 4.9 Mean 6.14.4 28.8

[0178] Toxicity of DSB was analyzed by incubating with PHA-stimulatedPBMC for 7 days at a range of concentrations, then determining cellviability using the XTT method. The 50% cytotoxic concentration was >30μM, corresponding to an in vitro therapeutic index of approximately5000.

Example 2

[0179] Anti-Viral Activity of DSB against Drug Resistant HIV-1 Isolates:

[0180] The activity of DSB was tested against a panel of HIV-1 isolatesresistant to approved drugs. These viruses were obtained from the NIHAIDS Research and Reference Reagent Program. Assays were performed usingvirus propagated in PBMCs with a p24 endpoint (above), or using cellline targets (MT-2 cells) and a cell killing endpoint. The MT-2 assayformat was as follows. Serial dilutions of DSB, or each approved drug,were prepared in 96 well plates. To each sample well was added mediacontaining MT-2 cells at 3×10⁵ cells/mL and virus inoculum at aconcentration necessary to result in 80% killing of the cell targets at5 days post-infection (PI). On day 5 post-infection, virus-induced cellkilling was determined by the XTT method and the inhibitory activity ofthe compound was determined.

[0181] Table 4 shows the potent anti-viral activity of DSB against apanel of drug-resistant HIV-1 isolates. The results were notsignificantly different from those obtained with the panel of wild-typeisolates (Table 3), demonstrating that DSB retains its activity againstvirus strains resistant to all of the major classes of approved drugs.TABLE 4 Table 4: Inhibitory activity (nM IC₅₀) of DSB against a panel ofdrug resistant HIV-1 isolates. Assays were done in fresh PBMC with a p24endpoint except for the NNRTI-resistant isolates that were performed inMT-2 cells with a cell viability (XTT) endpoint. * Fold Resistance.Virus Co-Receptor IC₅₀(nM) Isolate # Mutation(s) usage DSB AZTNevirapine Indinavir NRTI-resistant 1 K70R R5/X4 4.4 86.4 ND 9.8 T215Y/F(54X)* 2 K70R R5/X4 4.2 63.4 ND 6.1 T215Y/F (40X)  NNRTI-resistant 3Y181C X4 1.0  5.1 >3800 2.5 (>177X) 4 K103N X4 1.3  2.0   2630 4.5 Y181C  (122X) Protease-resistant 5 V82A X4 5.6 13.1 ND 39.7  (12X) 6 184V X45.5 14.4 ND 32.7  (10X) 7 L10R/M46I/ X4 12.9  3.5 ND 72.5 L63P/V82T/I84V (23X)

Example 3

[0182] DSB Inhibits HIV-1 Replication at a Late Step in the Virus LifeCycle

[0183] To distinguish the inhibitory activity of DSB against early andlate replication targets, a multinuclear activation of a galactosidaseindicator (MAGI) assay was used. In this assay, the targets are HeLacells stably expressing CD4, CXCR4, CCR5 and a reporter constructconsisting of the—galactosidase gene (modified to localize to thenucleus) driven by a truncated HIV-1 LTR. Infection of these cellsresults in expression of Tat that drives activation of theβ-galactosidase reporter gene. Expression of β-galactosidase in infectedcells is detected using the chromogenic substrate X-gal. As shown inTable 5, the entry inhibitor T-20, the NRTI AZT and the NNRTI nevirapinecaused significant reductions in β-galactosidase gene expression inHIV-1 infected MAGI cells due to their ability to disrupt early steps inviral replication that affect Tat protein expression. In contrast, theprotease inhibitor indinavir targets a late step in virus replication(following Tat expression) and does not prevent β-galactosidase geneexpression in this system. Similar results were obtained with DSB aswith indinavir, indicating that DSB blocks virus replication at a timepoint following the completion of proviral DNA integration and synthesisof the viral transactivating protein (Table 5). TABLE 5 Table 5: Effectof DSB and inhibitors of entry (the gp41 peptide T-20), RT (AZT andNevirapine) and protease (indinavir) on expression of b-galactosidase inHIV-1 infected MAGI cells. The DMSO control contained no drug. InhibitorDMSO T-20 AZT Nevirapine Indinavir DSB % Decrease (β- 0 98 82 85 10 12galactosidase expression)

[0184] Kanamoto et al. (Antimicrob. Agents Chemother., April;45(4):1225-30, (2002)) have also reported that DSB acts at a late stepin HIV replication. However, they reported that the compound inhibitsrelease of virus from chronically-infected cells. In contrast, our datausing a variety of experimental systems indicate that DSB does not havea significant effect on virus release (e.g. Example 6).

Example 4

[0185] DSB does not Inhibit HIV-1 Protease Activity

[0186] We had previously determined that DSB had no effect on HIV-1protease function using a cell-free fluorometric assay thatcharacterized enzyme activity by following the cleavage of a syntheticpeptide substrate. The results of these experiments indicated that atconcentrations up to 50 μg/mL that DSB had no effect on proteasefunction. As a result of the observation that DSB blocks virusreplication at a late step, studies were also performed using arecombinant form of the Gag protein, which is a more relevant systemthan the synthetic peptide substrate used in the initial assays. The useof the recombinant Gag protein as substrate resulted in a similarexperimental outcome. In these experiments DSB did not disruptprotease-mediated Gag protein processing at concentrations as high as 50μg/mL. In contrast, as expected, the protease inhibitor indinavirblocked Gag protein processing at 5 μg/mL (FIG. 1).

Example 5

[0187] DSB causes a defect in the final step of Gag processing (CA-SP1cleavage) that has been associated with viral maturation defects

[0188] In order to better define DSB's mechanism of action, a detailedexamination was undertaken of the virus produced from HIV-1-infectedcell lines treated with DSB. Briefly, H9 cells chronically infected withthe HIV-1_(IIIB) isolate were treated with DSB at 1 μg/mL for a periodof 48 hrs. Indinavir was used as a control. At the 48 hr time-point,spent media was removed and fresh media containing compound was added.At 24, 48 and 72 hrs post fresh compound addition, both cells andsupernatant were recovered for analysis. The level of virus in theculture supernatant was determined and western blots were used tocharacterize viral protein production in both cell-associated andcell-free virus. As observed in previous experiments, DSB did not causea significant reduction in the amount of virus, produced by chronicallyinfected H9 cells, however, there was a defect in Gag processing in bothcell-associated and cell-free virus. This defect took the form of anadditional band in the western blots corresponding to p25 (FIG. 2). Thisp25 band results from the incomplete processing of the capsid CA-SP1precursor. DSB treatment of HIV-2 and SIV chronically infected celllines exhibited normal Gag processing consistent with the observed lackof antiviral activity against these viruses. The Gag processing defectseen in the presence of DSB is completely distinct from that observedwith the protease inhibitor indinavir (FIG. 2). As discussed above,mutations at the p25 to p24 cleavage site that prevent processing areassociated with defects in viral maturation and infectivity (Wiegers K.et al., J. Virol. 72:2846-54 (1998)).

[0189] As previously discussed (C. T. Wild et al., XIV Int. AIDS Conf.Barcelona, Spain, Abstract MoPeA3030, (July 2002)), abnormal p25 to p24processing is also seen in other maturation budding defects. Theseinclude mutations in the Gag late domain (PTAP) or defects in TSG-101mediated viral assembly that disrupt budding (Garrus, J. E et al., Cell,107:55-65, (2001); Demirov, D. G. et al., J. Virology 76:105-117,(2002)). However, these mutations cause inhibition of virus release,while DSB treatment does not have a significant effect on virus release.The morphology of these maturation/budding mutants is also quitedistinct from that following DSB-treatment (see Example 6).

[0190] In addition, mutations that interfere with viral RNA dimerizationand lead to the production of immature virus with defective corestructures give a similar Gag processing phenotype (Liang, C. et al., J.Virology, 73:6147-6151, (1999)). However, in those cases RNAincorporation is inhibited and the morphology of particles released isdistinct from those following DSB treatment (see Example 6).

Example 6

[0191] DSB treatment effects HIV-1 maturation as determined by electronmicroscopy (EM)

[0192] It has been demonstrated that mutations in HIV-1 Gag that disruptp25 to p24 processing give rise to non-infectious viral particlescharacterized by an internal morphology distinct from normal virus(Wiegers K. et al., J. Virol. 72:2846-54 (1998)). To determine if virusgenerated in the presence of DSB exhibited this distinct rmorphology thefollowing experiment was carried out.

[0193] HeLa cells were transfected with HIV-1 infectious molecular clonepNL4-3 and treated as described previously with DSB. Followingtreatment, DSB-treated infected cells were fixed in glutaraldehyde andanalyzed by EM. The results of this analysis are shown in FIG. 3.

[0194] These results are consistent with a compound that disrupts p25 top24 processing which generates non-infectious morphologically aberrantviral particles.

[0195] 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB) is an exampleof a compound that disrupts p25 to p24 processing and potently inhibitsHIV-1 replication. However, this compound does not inhibit PR activity,and its action is specific for the p25 to p24 processing step, not othersteps in Gag processing. Furthermore, DSB treatment results in theaberrant HIV particle morphology described above.

Example 7

[0196] In vitro selection for HIV-1 isolates resistant to compounds thatdisrupt the processing of the viral Gag capsid (CA) protein from theCA-spacer peptide 1 protein precursor.

[0197] A series of experiments were performed to select for virusesresistant to inhibition by 3-O-(3′,3′-dimethylsuccinyl) betulinic acid(DSB), an inhibitor HIV-1 maturation. For each experiment, either NL4-3or RF virus isolate was used to infect two cell cultures. Followinginfection, one culture was maintained in growth medium containing DSB,while the other culture was maintained in parallel in growth mediumlacking DSB.

[0198] In one experiment, H9 cells that had been infected with RF viruswere maintained in the presence or absence of increasing concentrationsof DSB (0.05-1.6 μg/ml). The cells were passaged every 2-3 days with theaddition of fresh drug. Virus replication was monitored by p24 ELISAevery 7 days. At that time, DSB-treated cultures with high levels of p24were passaged by co-cultivation with fresh uninfected H9 cells at a 1:1ratio of cells in the presence of 1× or 2× the original concentration ofDSB. After 8 weeks of co-cultivation, cell-free virus was collected fromthe culture containing DSB at a concentration of 1.6 μg/ml and used toinfect fresh H9 cells. Every 7 days, virus from cultures containing highlevels of p24 was passaged by cell-free infection in the presence of 1×or 2× the original concentration of DSB. After 5 weeks of cell-freepassaging, virus from the culture containing 3.2 μg/ml DSB was collectedand used to infect MT-2 cells. Virus replication in the MT-2 cells, wasmonitored by observing syncytia formation microscopically. Every 1-3days, the cells were washed to remove input virus, and fresh drug wasadded to the culture under selection. Every 3-4 days, following theemergence of extensive syncytia in the culture under selection,supernatant from each culture was collected and passed through a 0.45 μmfilter to remove cell debris. This filtered virus supernatant was thenused to infect fresh MT-2 cells in the presence or absence of freshdrug. After 4 rounds of cell-free infection (approximately 2 weeks inculture), with the concentration of drug at 3.2 μg/ml, virus stocks werecollected and frozen for further analysis.

[0199] In a second experiment, a stock of virus derived from themolecular clone pNL4-3 (5.7×10⁴ TCID₅₀) was used to infect MT-2 cells(6×10⁶ cells) and cultures were maintained in the presence or absence ofPA-457 at a concentration of 1.6 μg/ml. Every 1-3 days, the cells werewashed to remove input virus, and fresh drug was added to the cultureunder selection. Virus replication was monitored by observing syncytiaformation microscopically. Every 3-7 days, following the emergence ofextensive syncytia in the culture under selection, supernatant from eachculture was collected and passed through a 0.45 μm filter to remove celldebris. This filtered virus supernatant was then used to infect freshMT-2 cells in the presence or absence of fresh drug. After 5 rounds ofcell-free infection, and every other round thereafter, the concentrationof drug was doubled. After 10 rounds of cell-free infection(approximately 7 weeks in culture), when the concentration of drugreached 12.8 μg/ml, virus stocks were collected and frozen for furtheranalysis.

Example 8

[0200] Characterization of HIV-1 isolates selected for resistance tocompounds that disrupt the processing of the viral Gag capsid (CA)protein from the CA-spacer peptide 1 protein precursor.

[0201] Virus stocks derived as described above were further analyzedboth phenotypically and genotypically to characterize the nature oftheir drug-resistance. The resistance of the viruses to3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) was determined invirus replication assays. Briefly, the virus stocks were first titeredin H9 cells by quantitating the levels of p24 (by ELISA) in cultures 8days after infection with serial 4-fold dilutions of virus. Virus inputwas then normalized for a second assay in which each virus is culturedfor 8 days in the presence of serial 4-fold dilutions of drug. The IC₅₀for each virus was determined as the dilution of drug that reduced thep24 endpoint level by 50% as compared to the no-drug control. Twoindependently derived virus stocks had IC₅₀ values greater than 1 μg/mlfor DSB, as compared to an IC₅₀ of 0.01 μg/ml for virus that had beencultured in parallel in the absence of drug.

[0202] To determine if the resistant viruses were able to escape theCA-SP1 cleavage defect caused by DSB in wild-type virus, stocks of eachvirus grown in either the presence or absence of drug were analyzed byWestern blot. Virus was pelleted through a 20% sucrose cushion fromfiltered culture supernatants that were collected 60 hr post-infectionand 18 hr after the cells had been washed and fresh drug added. Theviruses were lysed, and the amount of each virus was normalized byquantitating p24 levels in each sample. Western blot analysis of theviral proteins in each sample demonstrated that the drug-resistantviruses did not contain the CA-SP1 product in the presence of DSB,confirming that these viruses were resistant to the effects of the drugon this cleavage event.

[0203] Finally, to identify the genetic determinants of DSB resistance,the entire Gag and PR coding regions of the viral genomes were amplifiedby high-fidelity RT-PCR for sequencing. The viral RNA was purified fromeach virus lysate prepared as described above and digested with DNase toremove any contaminating DNA. The RT-PCR products were then gel-purifiedto remove any non-specific PCR products. Finally, both strands of theresulting DNA fragments were sequenced using overlapping a series ofprimers. Two amino acid mutations were identified that are independentlycapable of conferring resistance to DSB, an alanine to valinesubstitution in the Gag polyprotein at residue 364 in the NL4-3 isolateand at residue 366 in the RF isolate. These are the first and the thirdresidues, respectively, downstream of the CA-SP1 cleavage site (theN-terminus of SP1). Alanine is highly conserved at each of thesepositions throughout all HIV-1 clades in the database. Additionaldeterminants of resistance may be revealed by comparing the sequence ofSIV, which is resistant to DSB, in the region of its CA-SP1 cleavagesite (FIG. 10) to that of HIV-1 and by mutagenesis of the HIV-1 CA-SP1region.

[0204] Having now fully described this invention, it will be understoodto those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference in their entirety.

1 11 1 10 PRT HIV-1 MISC_FEATURE (4)..(4) Xaa can be either Val or Ile 1Lys Ala Arg Xaa Leu Ala Glu Ala Met Ser 1 5 10 2 10 PRT HIV-1 2 Lys AlaArg Val Leu Val Glu Ala Met Ser 1 5 10 3 10 PRT HIV-1 3 Lys Ala Arg IleLeu Ala Glu Val Met Ser 1 5 10 4 1823 DNA NL4-3 misc_feature(1365)..(1365) n is a, c, g, or t 4 atgggtgcga gagcgtcggt attaagcgggggagaattag ataaatggga aaaaattcgg 60 ttaaggccag ggggaaagaa acaatataaactaaaacata tagtatgggc aagcagggag 120 ctagaacgat tcgcagttaa tcctggccttttagagacat cagaaggctg tagacaaata 180 ctgggacagc tacaaccatc ccttcagacaggatcagaag aacttagatc attatataat 240 acaatagcag tcctctattg tgtgcatcaaaggatagatg taaaagacac caaggaagcc 300 ttagataaga tagaggaaga gcaaaacaaaagtaagaaaa aggcacagca agcagcagct 360 gacacaggaa acaacagcca ggtcagccaaaattacccta tagtgcagaa cctccagggg 420 caaatggtac atcaggccat atcacctagaactttaaatg catgggtaaa agtagtagaa 480 gagaaggctt tcagcccaga agtaatacccatgttttcag cattatcaga aggagccacc 540 ccacaagatt taaataccat gctaaacacagtggggggac atcaagcagc catgcaaatg 600 ttaaaagaga ccatcaatga ggaagctgcagaatgggata gattgcatcc agtgcatgca 660 gggcctattg caccaggcca gatgagagaaccaaggggaa gtgacatagc aggaactact 720 agtacccttc aggaacaaat aggatggatgacacataatc cacctatccc agtaggagaa 780 atctataaaa gatggataat cctgggattaaataaaatag taagaatgta tagccctacc 840 agcattctgg acataagaca aggaccaaaggaacccttta gagactatgt agaccgattc 900 tataaaactc taagagccga gcaagcttcacaagaggtaa aaaattggat gacagaaacc 960 ttgttggtcc aaaatgcgaa cccagattgtaagactattt taaaagcatt gggaccagga 1020 gcgacactag aagaaatgat gacagcatgtcagggagtgg ggggacccgg ccataaagca 1080 agagttttgg ttgaagcaat gagccaagtaacaaatccag ctaccataat gatacagaaa 1140 ggcaatttta ggaaccaaag aaagactgttaagtgtttca attgtggcaa agaagggcac 1200 atagccaaaa attgcagggc ccctaggaaaaagggctgtt ggaaatgtgg aaaggaagga 1260 caccaaatga aagattgtac tgagagacaggctaattttt tagggaagat ctggccttcc 1320 cacaagggaa ggccagggaa ttttcttcagagcagaccag agccnacagc cccaccagaa 1380 gagagcttca ggtttgggga agagacaacaactccctctc agaagcagga gccgatagac 1440 aaggaactgt atcctttagc ttccctcagatcactctttg gcagcgaccc ctcgtcacaa 1500 taaagatagg ggggcaatta aaggaagctctattagatac aggagcagat gatacagtat 1560 tagaagaaat gaatttgcca ggaagatggaaaccaaaaat gataggggga attggaggtt 1620 ttatcaaagt aagacagtat gatcagatactcatagaaat ctgcggacat aaagctatag 1680 gtacagtatt agtaggacct acacctgtcaacataattgg aagaaatctg ttgactcaga 1740 ttggctgcac tttaaatttt cccattagtcctattgagac tgtaccagta aaattaaagc 1800 caggaatgga tggcccaaaa gtt 1823 51820 DNA NL4-3 5 atgggtgcga gagcgtcggt attaagcggg ggagaattag ataaatgggaaaaaattcgg 60 ttaaggccag ggggaaagaa acaatataaa ctaaaacata tagtatgggcaagcagggag 120 ctagaacgat tcgcagttaa tcctggcctt ttagagacat cagaaggctgtagacaaata 180 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatcattatataat 240 acaatagcag tcctctattg tgtgcatcaa aggatagatg taaaagacaccaaggaagcc 300 ttagataaga tagaggaaga gcaaaacaaa agtaagaaaa aggcacagcaagcagcagct 360 gacacaggaa acaacagcca ggtcagccaa aattacccta tagtgcagaacctccagggg 420 caaatggtac atcaggccat atcacctaga actttaaatg catgggtaaaagtagtagaa 480 gagaaggctt tcagcccaga agtaataccc atgttttcag cattatcagaaggagccacc 540 ccacaagatt taaataccat gctaaacaca gtggggggac atcaagcagccatgcaaatg 600 ttaaaagaga ccatcaatga ggaagctgca gaatgggata gattgcatccagtgcaggca 660 gggcctattg caccaggcca gatgagagaa ccaaggggaa gtgacatagcaggaactact 720 agtacccttc aggaacaaat aggatggatg acacataatc cacctatcccagtaggagaa 780 atctataaaa gatggataat cctgggatta aataaaatag taagaatgtatagccctacc 840 agcattctgg acataagaca aggaccaaag gaacccttta gagactatgtagaccgattc 900 tataaaactc taagagccga gcaagcttca caagaggtaa aaaattggatgacagaaacc 960 ttgttggtcc aaaatgcgaa cccagattgt aagactattt taaaagcattgggaccagga 1020 gcgacactag aagaaatgat gacagcatgt cagggagtgg ggggacccggccataaagca 1080 agagttttgg ctgaagcaat gagccaagta acaaatccag ctaccataatgatacagaaa 1140 ggcaatttta ggaaccaaag aaagactgtt aagtgtttca attgtggcaaagaagggcac 1200 atagccaaaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtggaaaggaagga 1260 caccaaatga aagattgtac tgagagacag gctaattttt tagggaagatctggccttcc 1320 cacaagggaa ggccagggaa ttttcttcag agcagaccag agccaacagccccaccagaa 1380 gagagcttca ggtttgggga agagacaaca actccctctc agaagcaggagccgatagac 1440 aaggaactgt atcctttagc ttccctcaga tcactctttg gcagcgacccctcgtcacaa 1500 taaagatagg ggggcaatta aaggaagctc tattagatac aggagcagatgatacagtat 1560 tagaagaaat gaatttgcca ggaagatgga aaccaaaaat gatagggggaattggaggtt 1620 ttatcaaagt aagacagtat gatcagatac tcatagaaat ctgcggacataaagctatag 1680 gtacagtatt agtaggacct acacctgtca acataattgg aagaaatctgttgactcaga 1740 ttggctgcac tttaaatttt cccattagtc ctattgagac tgtaccagtaaaattaaagc 1800 caggaatgga tggcccaaag 1820 6 1982 DNA HIV-1RFmisc_feature (1961)..(1961) n ia a, c, g, or t 6 atgggtgcga gagcgtcagtattaagcggc ggaaaattag acaaatggga aaaaattcgg 60 ttaaggccag ggggaaagaaaagatataag ttaaaacata taatatgggc aagcagggag 120 ctagaacgat ttgctgtcaatcctggcctt ttagagacag cagagggctg tagacaaata 180 ctgggacagc tacaaccagcccttcagaca ggatcagaag aacttaaatc attatataat 240 gcagtagcaa ccctctattgtgtacatcaa aatatagagg taagagacac caaggaagct 300 ttagacaaga tagaggaagagcaaaacaaa agtaagaaaa aagcacagca agcagcagct 360 gacacaggaa acggcagccaggtcagccaa aattacccta tagtgcagaa ccttcagggg 420 caaatggtac atcaagccatatcacctaga actttaaatg catgggtaaa agtagtagaa 480 gagaaggctt ttagcccagaagtaataccc atgttttcag cattatcaga aggagccacc 540 ccacaagatt taaacaccatgctaaacaca gtggggggac atcaagcagc catgcaaatg 600 ttaaaagaga ctatcaatgaggaagctgca gaatgggata gattgcatcc agtgcaagca 660 gggcctattg caccaggccagatgagagaa ccaaggggaa gtgacatagc aggaaccact 720 agtacccttc aggaacaaataggatggatg acaaataatc cacctatccc agtaggagaa 780 atctataaaa ggtggataattctgggatta aataaaatag taagaatgta tagccccatc 840 agcattctgg acataagacaaggacctaag gaacccttta gagactatgt agaccggttc 900 tataaaactc taagagccgagcaagcttca caggatgtaa aaaattggat gacagaaacc 960 ttgctggtcc aaaatgcgaacccagattgt aaaactattt taaaagcatt gggaccagca 1020 gctacactag aagaaatgatgacagcatgt cagggagtag ggggacccag ccataaagca 1080 agaattttgg ctgaagtaatgagccaagta acaaattcag ctaccataat gctgcagaaa 1140 ggtaatttta gggaccaaagaaaaattgtt aagtgtttca actgtggcaa agtagggcac 1200 atagccaaaa attgcagggcccctaggaaa aagggctgtt ggaaatgtgg aaaggaagga 1260 caccaaatga aagattgcactactgaggga cgacaggcta attttttagg gaaaatctgg 1320 ccttcccaca agggaaggccagggaacttt cttcagagca gaccagagcc aacagcccca 1380 ccagaagaga gcttcaggtttggggaagag acaactccct ctcagaagca ggagaagata 1440 gacaaggaac tgtatcctttagcttccctc aaatcactct ttggcaacga cccatcgtca 1500 cagtaaagat aggggggcaattaaaggaag ctctattaga tacaggagca gatgatacag 1560 tattagaaga aatgaatttgccaggaaaat ggaaaccaaa aatgataggg ggaattggag 1620 gttttatcaa agtaaggcagtatgatcaaa tactcataga aatctgtgga cataaagcta 1680 taggtacagt attagtaggacctacacctg tcaacataat tggaagaaat ctgttgactc 1740 agattggttg cactttaaattttcccatta gtcctattga aactatacca gtaaaattaa 1800 agccaggaat ggatggcccaaaagttaaac aatggccatt gacagaggaa aaaataaaag 1860 cattgataga aatttgtacagaaatggaaa aggaaggaaa aatttcaaaa attgggcctg 1920 aaaatccata caatactccagtatttgcca taaagaaaaa ngacagtact aaatggagaa 1980 aa 1982 7 1904 DNAHIV-1RF 7 atgggtgcga gagcgtcagt attaagcggc ggaaaattag acaaatgggaaaaaattcgg 60 ttaaggccag ggggaaagaa aagatataag ttaaaacata taatatgggcaagcagggag 120 ctagaacgat ttgctgtcaa tcctggcctt ttagagacag cagagggctgtagacaaata 180 ctgggacagc tacaaccagc ccttcagaca ggatcagaag aacttaaatcattatataat 240 gcagtagcaa ccctctattg tgtacatcaa aatatagagg taagagacaccaaggaagct 300 ttagacaaga tagaggaaga gcaaaacaaa agtaagaaaa aagcacagcaagcagcagct 360 gacacaggaa acggcagcca ggtcagccaa aattacccta tagtgcagaaccttcagggg 420 caaatggtac atcaagccat atcacctaga actttaaatg catgggtaaaagtagtagaa 480 gagaaggctt ttagcccaga agtaataccc atgttttcag cattatcagaaggagccacc 540 ccacaagatt taaacaccat gctaaacaca gtggggggac atcaagcagccatgcaaatg 600 ttaaaagaga ctatcaatga ggaagctgca gaatgggata gattgcatccagtgcaagca 660 gggcctattg caccaggcca gatgagagaa ccaaggggaa gtgacatagcaggaaccact 720 agtacccttc aggaacaaat aggatggatg acaaataatc cacctatcccagtaggagaa 780 atctataaaa ggtggataat tctgggatta aataaaatag taagaatgtatagccccatc 840 agcattctgg acataagaca aggacctaag gaacccttta gagactatgtagaccggttc 900 tataaaactc taagagccga gcaagcttca caggatgtaa aaaattggatgacagaaacc 960 ttgctggtcc aaaatgcgaa cccagattgt aaaactattt taaaagcattgggaccagca 1020 gctacactag aagaaatgat gacagcatgt cagggagtag ggggacccagccataaagca 1080 agaattttgg ctgaagcaat gagccaagta acaaattcag ctaccataatgctgcagaaa 1140 ggtaatttta gggaccaaag aaaaattgtt aagtgtttca actgtggcaaagtagggcac 1200 atagccaaaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtggaaaggaagga 1260 caccaaatga aagattgcac tactgaggga cgacaggcta attttttagggaaaatctgg 1320 ccttcccaca agggaaggcc agggaacttt cttcagagca gaccagagccaacagcccca 1380 ccagaagaga gcttcaggtt tggggaagag acaactccct ctcagaagcaggagaagata 1440 gacaaggaac tgtatccttt agcttccctc aaatcactct ttggcaacgacccatcgtca 1500 cagtaaagat aggggggcaa ttaaaggaag ctctattaga tacaggagcagatgatacag 1560 tattagaaga aatgaatttg ccaggaaaat ggaaaccaaa aatgatagggggaattggag 1620 gttttatcaa agtaaggcag tatgatcaaa tactcataga aatctgtggacataaagcta 1680 taggtacagt attagtagga cctacacctg tcaacataat tggaagaaatctgttgactc 1740 agattggttg cactttaaat tttcccatta gtcctattga aactataccagtaaaattaa 1800 agccaggaat ggatggccca aaagttaaac aatggccatt gacagaggaaaaaataaaag 1860 cattgataga aatttgtaca gaaatggaaa aggaaggaaa aatt 1904 830 DNA HIV-1 8 aaagcaagag ttttggttga agcaatgagc 30 9 30 DNA HIV-1 9aaagcaagaa ttttggctga agtaatgagc 30 10 30 DNA HIV-1 misc_feature(10)..(10) n is either g or a 10 aaagcaagan ttttggctga agcaatgagc 30 1110 PRT SIV mac239 11 Lys Ala Arg Leu Met Ala Glu Ala Leu Lys 1 5 10

1. A method of treating HIV-1 infection in a patient by administering acompound that inhibits processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), but has no significant effect on other Gag processingsteps.
 2. The method of claim 1 wherein said inhibition does notsignificantly reduce the quantity of virions released from treatedinfected cells and/or has no significant effect on the amount of RNAincorporation into the released virions.
 3. The method of claim 1,wherein the compound inhibits the maturation of virions released fromtreated infected cells.
 4. The method of claim 1, wherein apreponderance of virions released from treated infected cells exhibitspherical, electron-dense cores that are acentric with respect to theviral particle, possess crescent-shaped electron-dense layers lying justinside the viral membrane, and have reduced or no infectivity.
 5. Themethod of claim 1, wherein the compound inhibits the interaction of HIVprotease with CA-SP1, which results in the inhibition of the processingof the viral Gag p25 protein (CA-SP1) to p24 (CA), but has nosignificant effect on other Gag processing steps.
 6. The method of claim1, wherein said compound binds to the viral Gag protein such thatinteraction of HIV protease with CA-SP1 is inhibited.
 7. The method ofclaim 1, wherein said compound binds near to or at the site of cleavageof the viral Gag p25 protein (CA-SP1) to p24 (CA), thereby inhibitingthe interaction of HIV protease with the CA-SP1 cleavage site andresulting in the inhibition of processing of p25 to p24.
 8. The methodof claim 1, wherein the HIV infecting said cells does not respond toother HIV therapies.
 9. The method of claim 1, wherein said patient isadministered said compound in combination with at least one anti-viralagent.
 10. The method of claim 9, wherein said anti-viral agent isselected from the group consisting of zidovudine, lamivudine,didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine,efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir,adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen,butylated hydroxytoluene; polymannoacetate, castanospermine; contracan;creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir,ganciclovir, dextran sulfate, D-penicillamine trisodiumphosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol,pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin,rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide,gp41-derived peptides, antibodies to CD4, soluble CD4, CD4-containingmolecules, CD4-IgG2, and combinations thereof.
 11. The method of claim1, wherein said patient is administered said compound in combinationwith an immunomodulating agent, anticancer agent, antibacterial agent,antifungal agent, or a combination thereof.
 12. The method of claim 1,wherein said compound is a dimethylsuccinyl betulinic acid ordimethylsuccinyl betulin derivative.
 13. The method of claim 12, whereinsaid compound is selected from the group consisting of3-O-(3′,3′-dimethylsuccinyl) betulinic acid,3-O-(3′,3′-dimethylsuccinyl) betulin, 3-O-(3′,3′-dimethylglutaryl)betulin, 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl) betulinic acid, (3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid,3-O-diglycolyl-dihydrobetulinic acid and combinations thereof.
 14. Themethod of claim 13, wherein said patient is administered said compoundin combination with at least one anti-viral agent.
 15. The method ofclaim 14, wherein said anti-viral agent is selected from the groupconsisting of zidovudine, lamivudine, didanosine, zalcitabine,stavudine, abacavir, nevirapine, delavirdine, efavirenz, saquinavir,ritonavir, indinavir, nelfinavir, amprenavir, adefovir, atazanavir,fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene;polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87,penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextransulfate, D-penicillamine trisodium phosphonoformate, fusidic acid,HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T,phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate,SK-818, suramin, UA001, enfuvirtide, gp41-derived peptides, antibodiesto CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, andcombinations thereof.
 16. The method of claim 13, wherein said patientis administered said compound in combination with an immunomodulatingagent, anti-cancer agent, antibacterial agent, an anti-fungal agent, orcombinations thereof.
 17. A method of treating human blood productscomprising contacting said blood products with a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but has nosignificant effect on other Gag processing steps.
 18. The method ofclaim 17 wherein said inhibition does not significantly reduce thequantity of virus released from treated cells and/or has no significanteffect on the amount of RNA incorporation into the released virions. 19.The method of claim 17, wherein the compound inhibits the maturation ofvirions released from treated infected cells.
 20. The method of claim17, wherein the preponderance of said virions released from treatedinfected cells exhibit spherical, electron-dense cores that are acentricwith respect to the virion, possess crescent-shaped electron-denselayers lying just inside the viral membrane, and have reduced or noinfectivity.
 21. The method of claim 17, wherein the compound inhibitsthe interaction of HIV protease with CA-SP1, which results in theinhibition of the processing of the viral Gag p25 protein (CA-SP1) top24 (CA), but has no significant effect on other Gag processing steps.22. The method of claim 17, wherein said compound binds near to or atthe site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA),thereby inhibiting the interaction of HIV protease with CA-SP1 andresulting in the inhibition of processing of p25 to p24.
 23. A methodfor identifying compounds that inhibit HIV-1 replication in cells of ananimal, comprising: (a) contacting a Gag protein comprising a CA-SP1cleavage site with a test compound; (b) adding a labeled substance thatselectively binds at or near the CA-SP1 cleavage site; and (c) measuringcompetition between the binding of the test compound and the labeledsubstance to the CA-SP1 cleavage site.
 24. The method of claim 23,wherein the compounds inhibit the interaction of HIV-1 protease with atarget site by binding to said target site.
 25. The method of claim 23,wherein the CA-SP1 is contained within a polypeptide fragment orrecombinant peptide.
 26. The method of claim 23, wherein the labeledsubstance is a labeled antibody specific for CA-SP1, and measuring thechange in the amount of labeled antibody bound to the protein in thepresence of test compound compared with a control.
 27. The method ofclaim 23, comprising measuring the change in the amount of labeled3-O-(3′,3′-dimethylsuccinyl) betulinic acid bound to the protein in thepresence of test compound, compared with a control, and wherein thelabeled substance is 3-O-(3′,3′-dimethylsuccinyl) betulinic acid. 28.The method according to claim 23 wherein the label is selected from thegroup consisting of an enzyme, fluorescent substance, chemiluminescentsubstance, horseradish peroxidase, alkaline phosphatase, biotin, avidin,electron dense substance, radioisotope and a combination thereof.
 29. Amethod for identifying compounds that inhibit HIV-1 replication in thecells of an animal comprising: (d) contacting a Gag protein comprising awild-type CA-SP1 cleavage site, with HIV-1 protease in the presence of atest compound; (e) separately, contacting a Gag protein comprising amutant CA-SP1 cleavage site or a protein comprising a alternativeprotease cleavage site with HIV-1 protease in the presence of the testcompound; and (f) comparing the amount of cleavage of the nativewild-type Gag protein to the amount of cleavage of the mutant Gagprotein or to amount of cleavage of the protein comprising analternative protease cleavage site.
 30. The method of claim 29, whereinthe wild-type CA-SP1 or mutant CA-SP1 or alternative protease cleavagesite is contained within a polypeptide fragment or recombinant peptide.31. The method of claim 29, wherein said Gag protein is labeled with afluorescent moiety and a fluorescence quenching moiety, each bound toopposite sides of the CA-SP1 cleavage site, and wherein said detectingcomprises measuring the signal from the fluorescent moiety.
 32. Themethod of claim 29, wherein said Gag protein is labeled with twofluorescent moieties, each bound to opposite sides of the CA-SP1cleavage site, and wherein said detecting comprises measuring thetransfer of fluorescent energy from one moiety to the other in thepresence of the test compound.
 33. The method of claim 29 wherein theeffect of the test compound on cleavage of the Gag protein is detectedby measuring the amount of a labeled antibody that is bound to SP1 orp24 (CA).
 34. The method of claim 33, wherein the labeled antibody thatbinds CA, or the antibody that binds SP1 is labeled with a moleculeselected from the group consisting of enzyme, fluorescent substance,chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, radioisotope, andcombinations thereof.
 35. A method for identifying compounds thatinhibit HIV-1 replication in cells of an animal comprising: (g)contacting a test compound with wild-type virus isolates and separatelywith virus isolates resistant to 3-O-(3′,3′-dimethylsuccinyl) betulinicacid; and (h) selecting test compounds that are more active against thewild-type virus isolate compared with virus isolates that are resistantto 3-O-(3′,3′-dimethylsuccinyl) betulinic acid.
 36. A method foridentifying compounds that inhibit HIV replication in the cells of ananimal, comprising: (i) contacting HIV-1 infected cells with a testcompound; and (j) thereafter lysing the infected cells or the releasedviral particles to form a lysate, and analyzing the lysate to determinewhether cleavage of the CA-SP1 protein has occurred.
 37. The method ofclaim 36, wherein said analyzing comprises measuring the presence orabsence of p25.
 38. The method of claim 36, wherein said analyzingcomprises performing a western blot of viral proteins and detecting p25using an antibody to p25.
 39. The method of claim 36, wherein saidanalyzing comprises performing a gel electrophoresis of viral proteinsand imaging of metabolically labeled proteins.
 40. The method of claim36, wherein said analyzing comprises using an antibody that selectivelybinds cleaved p24 (CA) or SP1 to distinguish p25 from p24.
 41. A methodfor identifying compounds that inhibit HIV-1 replication in the cells ofan animal comprising contacting HIV-1 infected cells with a testcompound and thereafter analyzing, wherein the virus particles releasedby the cells are analyzed by using transmission electron microscopy, forthe presence of spherical cores that are acentric with respect to theviral particle, and having crescent-shaped, electron-dense layers lyingjust inside the viral membrane.
 42. An isolated polynucleotidecomprising a sequence which encodes an amino acid sequence containing amutation in an HIV Gag p25 protein (CA SP1), said mutation resulting ina decrease in inhibition of processing of p25 (CA-SP1) to p24 (CA) by3-O-(3′,3′-dimethylsuccinyl) betulinic acid.
 43. The isolatedpolynucleotide of claim 42, wherein said decrease in inhibition ofprocessing of p25 is due to a decrease in inhibition of the interactionof HIV-1 protease with Gag.
 44. The isolated polynucleotide of claim 42,wherein said decrease in inhibition of processing of p25 is due to adecrease in the binding of 3-O-(3′,3′-dimethylsuccinyl) betulinic acidto Gag.
 45. The isolated polynucleotide of claim 42, wherein saiddecrease in inhibition of processing of p25 is due to a decrease in thebinding of DSB at or near the CA-SP1 cleavage site of Gag.
 46. Theisolated polynucleotide of claim 42, wherein said mutation is located inthe SP1 region of CA-SP1.
 47. The isolated polynucleotide of claim 42,wherein said mutation is located in the amino acid sequence KARV/ILAEAMS(SEQ ID NO: 1).
 48. The isolated polynucleotide of claim 42, whereinsaid mutation comprises an amino acid sequence that is selected from thegroup consisting of KARVLVEAMS (SEQ ID NO: 2) or KARILAEVMS (SEQ ID NO:3).
 49. The isolated polynucleotide of claim 42, comprising an aminoacid sequence encoded by a polynucleotide which is selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ IDNO:
 9. 50. The isolated polynucleotide of claim 42, having 95% identityto a polynucleotide selected from the group consisting of SEQ ID NO: 4,and SEQ ID NO:
 6. 51. The isolated polynucleotide of claim 42, having80% identity to a polynucleotide selected from the group consisting ofSEQ ID NO: 8 and SEQ ID NO:
 9. 52. The isolated polynucleotide of claim42, having 95% identity to a polynucleotide selected from the groupconsisting of SEQ NO: 5 and SEQ ID NO:
 7. 53. The isolatedpolynucleotide of claim 42, having 80% identity to a polynucleotide ofSEQ ID NO:
 10. 54. A vector comprising the isolated polynucleotide ofclaim
 42. 55. A host cell comprising the vector of claim
 54. 56. Amethod of producing a polypeptide comprising incubating the host cell ofclaim 55 in a medium and recovering the polypeptide from said medium.57. A virus comprising the isolated polynucleotide of claim
 42. 58. Aretrovirus comprising the isolated polynucleotide of claim
 42. 59. Theretrovirus of claim 58, selected from the group consisting of HIV-1,HIV-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenousavian retrovirus (EAV), mouse mammary tumor virus (MMTV), felineimmunodeficiency virus (FIV), or feline leukemia virus (FeLV).
 60. Theretrovirus of claim 59 which is HIV-1.
 61. A polypeptide containing amutation in an HIV CA-SP1 protein, said mutation which results in adecrease in inhibition of processing of p25 by3-O-(3′,3′-dimethylsuccinyl) betulinic acid.
 62. The polypeptide ofclaim 61, wherein said mutation is located in the SP1 region of SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO:
 10. 63. The polypeptide of claim 61,which is encoded by a polynucleotide selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO:
 9. 64. Thepolypeptide of claim 61, wherein said mutation comprises a sequence thatis selected from the group consisting of KARVLVEAMS (SEQ ID NO: 2) orKARILAEVMS (SEQ ID NO: 3).
 65. The polypeptide of claim 61, encoded byan isolated polynucleotide which hybridizes under highly stringentconditions to a polynucleotide selected from the group consisting of SEQID NO: 5, SEQ ID NO: 7, and
 10. 66. The polypeptide of claim 61, whereinsaid polypeptide is part of a chimeric or fusion protein.
 67. Anantibody which selectively binds an amino acid sequence containing amutation in an HIV CA-SP1 protein which results in a decrease in theinhibition of processing of p25 (CA-SP1) to p24 (CA) by3-O-(3′3′-dimethylsuccinyl) betulinic acid.
 68. The antibody of claim67, wherein said mutation is located in the SP1 region of CA-SP1. 69.The antibody of claim 68, wherein said mutation comprises a sequencethat is selected from the group consisting of KARVLVEAMS (SEQ ID NO: 2)or KARILAEVMS (SEQ ID NO: 3).
 70. The antibody of claim 67, whichselectively binds an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO:
 3. 71. An antibody thatselectively binds SP1 but not CA-SP1.
 72. An antibody that selectivelybinds CA but not CA-SP1.
 73. An antibody that selectively binds at ornear the CA-SP1 cleavage site.
 74. A compound identified by the methodof claim 23, 29, 35, 36, or 41, wherein the compound is not a compoundselected from the group consisting of 3-O-(3′,3′-dimethylsuccinyl)betulinic acid, 3-O-(3′,3′-dimethylsuccinyl) betulin,3-O-(3′,3′-dimethylglutaryl) betulin, 3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid, 3-O-(3′,3′-dimethylglutaryl) betulinic acid,(3′,3′-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof.75. A pharmaceutical composition comprising one or more compoundsaccording to claim 74, or a pharmaceutically acceptable salt, ester orprodrug thereof, and a pharmaceutically acceptable carrier.
 76. Apharmaceutical composition comprising a compound identified by themethod of claim, 23, 29, 35, 36, or 41, said composition furthercomprising an anti-viral agent.
 77. The pharmaceutical composition ofclaim 76 which comprises a dimethylsuccinyl betulinic acid ordimethylsuccinyl betulin derivative.
 78. The pharmaceutical compositionof claim 76, wherein said compound is selected from the group consistingof 3-O-(3′,3′-dimethylsuccinyl) betulinic acid,3-O-(3′,3′-dimethylsuccinyl) betulin, 3-O-(3′,3′-dimethylglutaryl)betulin, 3-O-(3′,3′-dimethylsuccinyl) dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl) betulinic acid, (3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid,3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof.
 79. Thepharmaceutical composition of claim 76, wherein said antiviral agent isselected from the group consisting of zidovudine, lamivudine,didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine,efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir,adefovir, atazanavir, hydroxyurea, AL-721, ampligen, butylatedhydroxytoluene; polymannoacetate, castanospermine; contracan; cremepharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir,ganciclovir, dextran sulfate, D-penicillamine trisodiumphosphonoformate, fusidic acid, HPA-23, eflomithine, nonoxynol,pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin,rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, andcombinations thereof.
 80. The pharmaceutical composition of claim 76,further comprising an immunomodulating agent, an anti-cancer agent, ananti-fungal agent, an anti-bacterial agent, or combinations thereof. 81.A method of determining if an individual is infected with HIV-1 that issusceptible to treatment by a compound that inhibits p25 processing thatinvolves taking blood from the patient, genotyping the viral RNA anddetermining whether the viral RNA contains mutations in the sequenceencoding the region of the CA-SP1.